Lipids and compositions for the delivery of therapeutics

ABSTRACT

The present invention provides lipids that are advantageously used in lipid particles for the in vivo delivery of therapeutic agents to cells. In particular, the invention provides lipids having the following structure 
                         
wherein:
 
R 1  and R 2  are each independently for each occurrence optionally substituted C 10 -C 30  alkyl, optionally substituted C 10 -C 30  alkenyl, optionally substituted C 10 -C 30  alkynyl, optionally substituted C 10 -C 30  acyl, or -linker-ligand; R 3  is H, optionally substituted C 1 -C 10  alkyl, optionally substituted C 2 -C 10  alkenyl, optionally substituted C 2 -C 10  alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw 100-40 K), optionally substituted mPEG (mw 120-40 K), heteroaryl, heterocycle, or linker-ligand; and E is C(O)O or OC(O).

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.13/128,283, which is the National Stage of International PatentApplication No. PCT/US09/63927, filed Nov. 10, 2009, which claimspriority to U.S. Application No. 61/113,179, filed Nov. 10, 2008; U.S.Application No. 61/154,350, filed Feb. 20, 2009; U.S. Application No.61/171,439, filed Apr. 21, 2009; U.S. Application No. 61/185,438, filedJun. 9, 2009; U.S. Application No. 61/225,898, filed Jul. 15, 2009; andU.S. Application No. 61/234,098, filed Aug. 14, 2009, the contents ofeach of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under HHSN266200600012Cawarded by the National Institute of Allergy and Infectious Diseases.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 8, 2015, isnamed 08050.007US2_SL.txt and is 22,205 bytes in size.

BACKGROUND

Technical Field

The present invention relates to the field of therapeutic agent deliveryusing lipid particles. In particular, the present invention providescationic lipids and lipid particles comprising these lipids, which areadvantageous for the in vivo delivery of nucleic acids, as well asnucleic acid-lipid particle compositions suitable for in vivotherapeutic use. Additionally, the present invention provides methods ofmaking these compositions, as well as methods of introducing nucleicacids into cells using these compositions, e.g., for the treatment ofvarious disease conditions.

Description of the Related Art

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids,immune stimulating nucleic acids, antisense, antagomir, antimir,microRNA mimic, supermir, U1 adaptor, and aptamer. These nucleic acidsact via a variety of mechanisms. In the case of siRNA or miRNA, thesenucleic acids can down-regulate intracellular levels of specificproteins through a process termed RNA interference (RNAi). Followingintroduction of siRNA or miRNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the siRNA or miRNA is displaced from the RISC complexproviding a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound siRNA or miRNA. Having boundthe complementary mRNA the RISC complex cleaves the mRNA and releasesthe cleaved strands. RNAi can provide down-regulation of specificproteins by targeting specific destruction of the corresponding mRNAthat encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, since siRNAand miRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as the freesiRNA or miRNA. These double-stranded constructs can be stabilized byincorporation of chemically modified nucleotide linkers within themolecule, for example, phosphothioate groups. However, these chemicalmodifications provide only limited protection from nuclease digestionand may decrease the activity of the construct. Intracellular deliveryof siRNA or miRNA can be facilitated by use of carrier systems such aspolymers, cationic liposomes or by chemical modification of theconstruct, for example by the covalent attachment of cholesterolmolecules. However, improved delivery systems are required to increasethe potency of siRNA and miRNA molecules and reduce or eliminate therequirement for chemical modification.

Antisense oligonucleotides and ribozymes can also inhibit mRNAtranslation into protein. In the case of antisense constructs, thesesingle stranded deoxynucleic acids have a complementary sequence to thatof the target protein mRNA and can bind to the mRNA by Watson-Crick basepairing. This binding either prevents translation of the target mRNAand/or triggers RNase H degradation of the mRNA transcripts.Consequently, antisense oligonucleotides have tremendous potential forspecificity of action (i.e., down-regulation of a specificdisease-related protein). To date, these compounds have shown promise inseveral in vitro and in vivo models, including models of inflammatorydisease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech.14:376-387 (1996)). Antisense can also affect cellular activity byhybridizing specifically with chromosomal DNA. Advanced human clinicalassessments of several antisense drugs are currently underway. Targetsfor these drugs include the bcl2 and apolipoprotein B genes and mRNAproducts.

Immune-stimulating nucleic acids include deoxyribonucleic acids andribonucleic acids. In the case of deoxyribonucleic acids, certainsequences or motifs have been shown to illicit immune stimulation inmammals. These sequences or motifs include the CpG motif,pyrimidine-rich sequences and palindromic sequences. It is believed thatthe CpG motif in deoxyribonucleic acids is specifically recognized by anendosomal receptor, toll-like receptor 9 (TLR-9), which then triggersboth the innate and acquired immune stimulation pathway. Certain immunestimulating ribonucleic acid sequences have also been reported. It isbelieved that these RNA sequences trigger immune activation by bindingto toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,double-stranded RNA is also reported to be immune stimulating and isbelieve to activate via binding to TLR-3.

One well known problem with the use of therapeutic nucleic acids relatesto the stability of the phosphodiester internucleotide linkage and thesusceptibility of this linker to nucleases. The presence of exonucleasesand endonucleases in serum results in the rapid digestion of nucleicacids possessing phosphodiester linkers and, hence, therapeutic nucleicacids can have very short half-lives in the presence of serum or withincells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); andThierry, A. R., et al., pp 147-161 in Gene Regulation: Biology ofAntisense RNA and DNA (Eds. Erickson, R P and Izant, J G; Raven Press,NY (1992)). Therapeutic nucleic acid being currently being developed donot employ the basic phosphodiester chemistry found in natural nucleicacids, because of these and other known problems.

This problem has been partially overcome by chemical modifications thatreduce serum or intracellular degradation. Modifications have beentested at the internucleotide phosphodiester bridge (e.g., usingphosphorothioate, methylphosphonate or phosphoramidate linkages), at thenucleotide base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,2′-modified sugars) (Uhlmann E., et al. Antisense: ChemicalModifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic PressInc. (1997)). Others have attempted to improve stability using 2′-5′sugar linkages (see, e.g., U.S. Pat. No. 5,532,130). Other changes havebeen attempted. However, none of these solutions have proven entirelysatisfactory, and in vivo free therapeutic nucleic acids still have onlylimited efficacy.

In addition, as noted above relating to siRNA and miRNA, problems remainwith the limited ability of therapeutic nucleic acids to cross cellularmembranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082(1994)) and in the problems associated with systemic toxicity, such ascomplement-mediated anaphylaxis, altered coagulatory properties, andcytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206(1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. In Zelphati, O and Szoka, F. C., J. Contr.Rel. 41:99-119 (1996), the authors refer to the use of anionic(conventional) liposomes, pH sensitive liposomes, immunoliposomes,fusogenic liposomes, and cationic lipid/antisense aggregates. SimilarlysiRNA has been administered systemically in cationic liposomes, andthese nucleic acid-lipid particles have been reported to provideimproved down-regulation of target proteins in mammals includingnon-human primates (Zimmermann et al., Nature 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these lipid-nucleic acid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

BRIEF SUMMARY

The present invention provides novel cationic lipids, as well as lipidparticles comprising the same. These lipid particles may furthercomprise an active agent and be used according to related methods of theinvention to deliver the active agent to a cell.

In one aspect, the invention provides lipids having the structure

salts or isomers thereof, wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl, or -linker-ligand;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40 K),optionally substituted mPEG (mw 120-40 K), heteroaryl, heterocycle, orlinker-ligand; and

E is C(O)O or OC(O).

In another aspect, the invention provides a lipid particle comprisingthe lipids of the present invention. In certain embodiments, the lipidparticle further comprises a neutral lipid and a lipid capable ofreducing particle aggregation. In one embodiment, the lipid particleconsists essentially of (i) at least one lipid of the present invention;(ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii)sterol, e.g. cholesterol; and (iv) peg-lipid, e.g. PEG-DMG or PEG-DMA,in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid. In one embodiment, the lipid ofthe present invention is optically pure.

In additional related embodiments, the present invention includes lipidparticles of the invention that further comprise therapeutic agent. Inone embodiment, the therapeutic agent is a nucleic acid. In oneembodiment, the nucleic acid is a plasmid, an immunostimulatoryoligonucleotide, a single stranded oligonucleotide, e.g. an antisenseoligonucleotide, an antagomir; a double stranded oligonucleotide, e.g. asiRNA; an aptamer or a ribozyme.

In yet another related embodiment, the present invention includes apharmaceutical composition comprising a lipid particle of the presentinvention and a pharmaceutically acceptable excipient, carrier ofdiluent.

The present invention further includes, in other related embodiments, amethod of modulating the expression of a target gene in a cell, themethod comprising providing to a cell a lipid particle or pharmaceuticalcomposition of the present invention. The target gene can be a wild typegene. In another embodiment, the target gene contains one or moremutations. In a particular embodiment, the method comprises specificallymodulating expression of a target gene containing one or more mutations.In particular embodiments, the lipid particle comprises a therapeuticagent selected from an immunostimulatory oligonucleotide, a singlestranded oligonucleotide, e.g. an antisense oligonucleotide, anantagomir; a double stranded oligonucleotide, e.g. a siRNA, an aptamer,a ribozyme. In one embodiment, the nucleic acid is plasmid that encodesa siRNA, an antisense oligonucleotide, an aptamer or a ribozyme.

In one aspect of the invention, the target gene is selected from thegroup consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV,PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene,MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene,JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene,Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene,PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, SORT1gene, XBP1 gene, topoisomerase I gene, topoisomerase II alpha gene, p73gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene,caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumorsuppressor genes, p53 tumor suppressor gene, and combinations thereof.

In another embodiment, the nucleic acid is a plasmid that encodes apolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In yet a further related embodiment, the present invention includes amethod of treating a disease or disorder characterized by overexpressionof a polypeptide in a subject, comprising providing to the subject alipid particle or pharmaceutical composition of the present invention,wherein the therapeutic agent is selected from an siRNA, a microRNA, anantisense oligonucleotide, and a plasmid capable of expressing an siRNA,a microRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In a further embodiment, the present invention includes a method ofinducing an immune response in a subject, comprising providing to thesubject a pharmaceutical composition of the present invention, whereinthe therapeutic agent is an immunostimulatory oligonucleotide. Inparticular embodiments, the pharmaceutical composition is provided tothe patient in combination with a vaccine or antigen.

In a related embodiment, the present invention includes a vaccinecomprising the lipid particle of the present invention and an antigenassociated with a disease or pathogen. In one embodiment, the lipidparticle comprises an immunostimulatory nucleic acid or oligonucleotide.In a particular embodiment, the antigen is a tumor antigen. In anotherembodiment, the antigen is a viral antigen, a bacterial antigen, or aparasitic antigen.

The present invention further includes methods of preparing the lipidparticles and pharmaceutical compositions of the present invention, aswell as kits useful in the preparation of these lipid particle andpharmaceutical compositions.

In another aspect, the invention provides a method of evaluating acomposition that includes an agent, e.g. a therapeutic agent ordiagnostic agent, and a lipid of the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Schematic representation of an optically pure lipid withconjugated targeting ligands.

FIG. 2. Schematic representation of features of the lipids of thepresent invention.

FIG. 3. shows a table depicting the EC50 and pKa values of exemplarylipids tested using method described in the Examples.

DETAILED DESCRIPTION

The present invention is based, in part, upon the discovery of cationiclipids that provide advantages when used in lipid particles for the invivo delivery of a therapeutic agent. In particular, as illustrated bythe accompanying Examples, the present invention provides nucleicacid-lipid particle compositions comprising a cationic lipid accordingto the present invention. In some embodiments, a composition describedherein provides increased activity of the nucleic acid and/or improvedtolerability of the compositions in vivo, which can result in asignificant increase in therapeutic index as compared to lipid-nucleicacid particle compositions previously described. Additionallycompositions and methods of use are disclosed that can provide foramelioration of the toxicity observed with certain therapeutic nucleicacid-lipid particles.

In certain embodiments, the present invention specifically provides forimproved compositions for the delivery of siRNA molecules. It is shownherein that these compositions are effective in down-regulating theprotein levels and/or mRNA levels of target proteins. Furthermore, it isshown that the activity of these improved compositions is dependent onthe presence of a certain cationic lipids and that the molar ratio ofcationic lipid in the formulation can influence activity.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of associated orencapsulated therapeutic agents to cells, both in vitro and in vivo.Accordingly, the present invention provides methods of treating diseasesor disorders in a subject in need thereof, by contacting the subjectwith a lipid particle of the present invention associated with asuitable therapeutic agent.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,siRNA molecules and plasmids. Therefore, the lipid particles andcompositions of the present invention may be used to modulate theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle of the present inventionassociated with a nucleic acid that reduces target gene expression(e.g., an siRNA) or a nucleic acid that may be used to increaseexpression of a desired protein (e.g., a plasmid encoding the desiredprotein).

Various exemplary embodiments of the cationic lipids of the presentinvention, as well as lipid particles and compositions comprising thesame, and their use to deliver therapeutic agents and modulate gene andprotein expression are described in further detail below.

Lipids

The present invention provides novel lipids having certain designfeatures. As shown in FIG. 2, the lipid design features include at leastone of the following: a head group with varying pKa, a cationic, 1°, 2°and 3°, monoamine, Di and triamine, Oligoamine/polyamine, a low pKa headgroups—imidazoles and pyridine, guanidinium, anionic, zwitterionic andhydrophobic tails can include symmetric and/or unsymmetric chains, longand shorter, saturated and unsaturated chain the back bone includesBackbone glyceride and other acyclic analogs, cyclic, spiro, bicyclicand polycyclic linkages with ethers, esters, phosphate and analogs,sulfonate and analogs, disulfides, pH sensitive linkages like acetalsand ketals, imines and hydrazones, and oximes.

The present invention provides novel lipids that are advantageously usedin lipid particles of the present invention for the in vivo delivery oftherapeutic agents to cells, including lipids having the followingstructure In one aspect, the lipid is a compound of formula XXXIII,

salts or isomers thereof, wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl, or -linker-ligand;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40 K),optionally substituted mPEG (mw 120-40 K), heteroaryl, heterocycle, orlinker-ligand; and

E is C(O)O or OC(O).

In one embodiment, R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl.

In another embodiment, R₃ is H, optionally substituted C₁-C₁₀ alkyl,optionally substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted alkylheterocycle, optionally substitutedheterocyclealkyl, optionally substituted alkylphosphate, optionallysubstituted phosphoalkyl, optionally substituted alkylphosphorothioate,optionally substituted phosphorothioalkyl, optionally substitutedalkylphosphorodithioate, optionally substituted phosphorodithioalkyl,optionally substituted alkylphosphonate, optionally substitutedphosphonoalkyl, optionally substituted amino, optionally substitutedalkylamino, optionally substituted di(alkyl)amino, optionallysubstituted aminoalkyl, optionally substituted alkylaminoalkyl,optionally substituted di(alkyl)aminoalkyl, optionally substitutedhydroxyalkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40 K), optionally substitutedheteroaryl, optionally substituted heterocycle, or linker-ligand.

In one embodiment, where the lipid is a compound of formula XXXIII,provided that when E is C(O)O and R³ is

R¹ and R² are not both linoleyl.

In one embodiment, the lipid is a compound of formula XXXIII, wherein R₃is H, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls,ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls,ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw100-40 K), optionally substituted mPEG (mw 120-40 K), heteroaryl,heterocycle, or linker-ligand.

In yet another embodiment, the lipid is a compound of formula XXXIII,wherein R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkynyl,optionally substituted C₁₀-C₃₀ acyl, or -linker-ligand.

In one aspect, the invention features a lipid of formula XXXVIII:

salts or isomers thereof,

wherein

E is C(O)O or OC(O);

R₁ and R₂ and R_(x) are each independently for each occurrence H,optionally substituted C₁-C₁₀ alkyl, optionally substituted C₁₀-C₃₀alkyl, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀ acyl, or linker-ligand,provided that at least one of R₁, R₂ and R_(x) is not H;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40 K),optionally substituted mPEG (mw 120-40 K), heteroaryl, heterocycle, orlinker-ligand;

n is 0, 1, 2, or 3.

In one embodiment, where the lipid is a compound of formula XXXVIII,provided that when E is C(O)O, R³ is

and one of R₁, R₂, or R_(x) is H, then the remaining of R₁, R₂, or R_(x)are not both linoleyl.

In some embodiments, each of R₁ and R₂ is independently for eachoccurrence optionally substituted C₁₀-C₃₀ alkyl, optionally substitutedC₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ acyl, or linker-ligand.

In some embodiments, R_(x) is H or optionally substituted C₁-C₁₀ alkyl.

In some embodiments, R_(x) is optionally substituted C₁₀-C₃₀ alkyl,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkynyl, optionally substituted C₁₀-C₃₀ acyl, or linker-ligand.

In one embodiment, R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl,or -linker-ligand.

In one embodiment, R₃ is independently for each occurrence H, optionallysubstituted C₁-C₁₀ alkyl, optionally substituted C₂-C₁₀ alkenyl,optionally substituted C₂-C₁₀ alkynyl, optionally substitutedalkylheterocycle, optionally substituted heterocyclealkyl, optionallysubstituted alkylphosphate, optionally substituted phosphoalkyl,optionally substituted alkylphosphorothioate, optionally substitutedphosphorothioalkyl, optionally substituted alkylphosphorodithioate,optionally substituted phosphorodithioalkyl, optionally substitutedalkylphosphonate, optionally substituted phosphonoalkyl, optionallysubstituted amino, optionally substituted alkylamino, optionallysubstituted di(alkyl)amino, optionally substituted aminoalkyl,optionally substituted alkylaminoalkyl, optionally substituteddi(alkyl)aminoalkyl, optionally substituted hydroxyalkyl, optionallysubstituted polyethylene glycol (PEG, mw 100-40 K), optionallysubstituted mPEG (mw 120-40 K), optionally substituted heteroaryl, oroptionally substituted heterocycle, or linker-ligand.

In one embodiment, E is —C(O)O— or —OC(O)—.

In one embodiment, Z′ is —O—, —S—, —N(Q)-, or alkylene.

In some circumstances, R₃ is ω-aminoalkyl, ω-(substituted)aminoalkyl,ω-phosphoalkyl, or ω-thiophosphoalkyl; each of which is optionallysubstituted. Examples of ω-(substituted)aminoalkyl groups include2-(dimethylamino)ethyl, 3-(diisopropylamino)propyl, or3-(N-ethyl-N-isopropylamino)-1-methylpropyl.

It has been found that cationic lipids comprising unsaturated alkylchains are particularly useful for forming lipid nucleic acid particleswith increased membrane fluidity. In one embodiment, at least one of R₁or R₂ comprises at least one, at least two or at least three sites ofunsaturation, e.g. double bond or triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one, at leasttwo or at least three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise at least one, at least two orat least three sites of unsaturation.

In one embodiment, R₁ and R₂ comprise different numbers of unsaturation,e.g., one of R₁ and R₂ has one site of unsaturation and the other hastwo or three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise the same number ofunsaturation sites.

In one embodiment, R₁ and R₂ comprise different types of unsaturation,e.g. unsaturation in one of R₁ and R₂ is double bond and in the otherunsaturation is triple bond.

In one embodiment, R₁ and R₂ both comprise the same type ofunsaturation, e.g. double bond or triple bond.

In one embodiment, at least one of R₁ or R₂ comprises at least onedouble bond and at least one triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one doublebond and at least one triple bond.

In one embodiment, R₁ and R₂ both comprise at least one double bond andat least one triple bond.

In one embodiment, R₁ and R₂ are both same, e.g. R₁ and R₂ are bothlinoleyl (C18) or R₁ and R₂ are both heptadeca-9-enyl.

In one embodiment, R₁ and R₂ are different from each other.

In one embodiment, at least one of R₁ and R₂ is cholesterol.

In one embodiment, one of R₁ and R₂ is -linker-ligand.

In one embodiment, one of R₁ and R₂ is -linker-ligand and ligand is alipophile.

In one embodiment, at least one of R₁ or R₂ comprises at least one CH₂group with one or both H replaced by F, e.g. CHF or CF₂. In oneembodiment, both R₁ and R₂ comprise at least one CH₂ group with one ortwo H replaced by F, e.g. CHF or CF₂.

In one embodiment, only one of R₁ and R₂ comprises at least one CH₂group with one or both H replaced by F.

In one embodiment, at least one of R₁ or R₂ terminates in CH₂F, CHF₂ orCF₃. In one embodiment, both R₁ and R₂ terminate in CH₂F, CHF₂ or CF₃.

In one embodiment, at least one of R₁ or R₂ is—(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is—(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In one embodiment, R₃ is chosen from a group consisting of methyl,ethyl, polyamine, —(CH₂)_(h)-heteroaryl, —(CH₂)_(h)—N(Q)₂, —O—N(Q)₂,—(CH₂)_(h)—Z′—(CH₂)_(h)-heteroaryl, linker-ligand,—(CH₂)_(h)-heterocycle, and —(CH₂)_(h)—Z″—(CH₂)_(h)-heterocycle, whereineach h is independently 0-13 and Z″ is O, S or N(Q).

In one embodiment, when Z is C(R₃), at least one R₃ is ω-aminoalkyl orω-(substituted)aminoalkyl.

In one embodiment, when Z′ is O, S or alkyl, at least one R₃ isω-aminoalkyl or ω-(substituted)aminoalkyl.

In one embodiment, Q is linker-ligand.

In one embodiment, ligand is fusogenic peptide.

In one embodiment, the lipid is a racemic mixture.

In one embodiment, the lipid is enriched in one diastereomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%diastereomeric excess.

In one embodiment, the lipid is enriched in one enantiomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%enantiomer excess.

In one embodiment, the lipid is chirally pure, e.g. is a single opticalisomer.

In one embodiment, the lipid is enriched for one optical isomer.

Where a double bond is present (e.g., a carbon-carbon double bond orcarbon-nitrogen double bond), there can be isomerism in theconfiguration about the double bond (i.e. cis/trans or E/Z isomerism).Where the configuration of a double bond is illustrated in a chemicalstructure, it is understood that the corresponding isomer can also bepresent. The amount of isomer present can vary, depending on therelative stabilities of the isomers and the energy required to convertbetween the isomers. Accordingly, some double bonds are, for practicalpurposes, present in only a single configuration, whereas others (e.g.,where the relative stabilities are similar and the energy of conversionlow) may be present as inseparable equilibrium mixture ofconfigurations.

The present invention comprises of synthesis of lipids described hereinin racemic as well as in optically pure form.

In one embodiment, the cationic lipid is chosen from a group consistingof lipids shown in Table 1 below.

TABLE 1 Some cationic lipids of the present invention.

Although not all diasteromers for a lipid are shown, one aspect of thepresent invention is to provide all diastereomers and as such chirallypure and diastereomerically enriched lipids are also part of thisinvention.

In one embodiment, R₃ is -linker-ligand.

In particular embodiments, the lipids of the present invention arecationic lipids. As used herein, the term “cationic lipid” is meant toinclude those lipids having one or two fatty acid or fatty alkyl chainsand an amino head group (including an alkylamino or dialkylamino group)that may be protonated to form a cationic lipid at physiological pH. Insome embodiments, a cationic lipid is referred to as an “amino lipid.”

Other cationic lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R₁and R₂ are both long chain alkyl or acyl groups, they can be the same ordifferent. In general, lipids (e.g., a cationic lipid) having lesssaturated acyl chains are more easily sized, particularly when thecomplexes are sized below about 0.3 microns, for purposes of filtersterilization. Cationic lipids containing unsaturated fatty acids withcarbon chain lengths in the range of C₁₀ to C₂₀ are typical. Otherscaffolds can also be used to separate the amino group (e.g., the aminogroup of the cationic lipid) and the fatty acid or fatty alkyl portionof the cationic lipid. Suitable scaffolds are known to those of skill inthe art.

In certain embodiments, cationic lipids of the present invention have atleast one protonatable or deprotonatable group, such that the lipid ispositively charged at a pH at or below physiological pH (e.g. pH 7.4),and neutral at a second pH, preferably at or above physiological pH.Such lipids are also referred to as cationic lipids. It will, of course,be understood that the addition or removal of protons as a function ofpH is an equilibrium process, and that the reference to a charged or aneutral lipid refers to the nature of the predominant species and doesnot require that all of the lipid be present in the charged or neutralform. Lipids that have more than one protonatable or deprotonatablegroup, or which are zwiterrionic, are not excluded from use in theinvention.

In certain embodiments, protonatable lipids (i.e., cationic lipids)according to the invention have a pKa of the protonatable group in therange of about 4 to about 11. Typically, lipids will have a pKa of about4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and6.8, when incorporated into lipid particles. Such lipids will becationic at a lower pH formulation stage, while particles will belargely (though not completely) surface neutralized at physiological pHaround pH 7.4. One of the benefits of a pKa in the range of betweenabout 4 and 7 is that at least some nucleic acid associated with theoutside surface of the particle will lose its electrostatic interactionat physiological pH and be removed by simple dialysis; thus greatlyreducing the particle's susceptibility to clearance. pKa measurements oflipids within lipid particles can be performed, for example, by usingthe fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS),using methods described in Cullis et al., (1986) Chem Phys Lipids 40,127-144.

In one embodiment, the formulations of the invention are entrapped by atleast 75%, at least 80% or at least 90%.

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below. Suitable apolipoproteinsinclude, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V andApoE, and active polymorphic forms, isoforms, variants and mutants aswell as fragments or truncated forms thereof. In certain embodiments,the apolipoprotein is a thiol containing apolipoprotein. “Thiolcontaining apolipoprotein” refers to an apolipoprotein, variant,fragment or isoform that contains at least one cysteine residue. Themost common thiol containing apolipoproteins are ApoA-I Milano(ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) which contain one cysteineresidue (Jia et al., 2002, Biochem. Biophys. Res. Comm. 297: 206-13;Bielicki and Oda, 2002, Biochemistry 41: 2089-96). ApoA-II, ApoE2 andApoE3 are also thiol containing apolipoproteins. Isolated ApoE and/oractive fragments and polypeptide analogues thereof, includingrecombinantly produced forms thereof, are described in U.S. Pat. Nos.5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189; 5,168,045;5,116,739; the disclosures of which are herein incorporated byreference. ApoE3 is disclosed in Weisgraber, et al., “Human E apoproteinheterogeneity: cysteine-arginine interchanges in the amino acid sequenceof the apo-E isoforms,” J. Biol. Chem. (1981) 256: 9077-9083; and Rall,et al., “Structural basis for receptor binding heterogeneity ofapolipoprotein E from type III hyperlipoproteinemic subjects,” Proc.Nat. Acad. Sci. (1982) 79: 4696-4700. See also GenBank accession numberK00396.

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IMilano (Klon et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al.,1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be anyof those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166,and 5,840,688, the contents of which are incorporated herein byreference in their entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et. al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogenous mixtures of apolipoproteinscan comprise, for example, a mixture of an apolipoprotein from an animalsource and an apolipoprotein from a semi-synthetic source. In certainembodiments, a heterogenous mixture can comprise, for example, a mixtureof ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneousmixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-IParis. Suitable mixtures for use in the methods and compositions of theinvention will be apparent to one of skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotien can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

Lipid Particles

The present invention also provides lipid particles comprising one ormore of the cationic lipids described above. Lipid particles include,but are not limited to, liposomes. As used herein, a liposome is astructure having lipid-containing membranes enclosing an aqueousinterior. Liposomes may have one or more lipid membranes. The inventioncontemplates both single-layered liposomes, which are referred to asunilamellar, and multi-layered liposomes, which are referred to asmultilamellar. When complexed with nucleic acids, lipid particles mayalso be lipoplexes, which are composed of cationic lipid bilayerssandwiched between DNA layers, as described, e.g., in Feigner,Scientific American.

The lipid particles of the present invention may further comprise one ormore additional lipids and/or other components such as cholesterol.Other lipids may be included in the liposome compositions of the presentinvention for a variety of purposes, such as to prevent lipid oxidationor to attach ligands onto the liposome surface. Any of a number oflipids may be present in liposomes of the present invention, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described below.

Additional components that may be present in a lipid particle of thepresent invention include bilayer stabilizing components such aspolyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides,proteins, detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

In particular embodiments, the lipid particles include one or more of asecond amino lipid or cationic lipid, a neutral lipid, a sterol, and alipid selected to reduce aggregation of lipid particles duringformation, which may result from steric stabilization of particles whichprevents charge-induced aggregation during formation.

Examples of lipids that reduce aggregation of particles during formationinclude polyethylene glycol (PEG)-modified lipids, monosialogangliosideGm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No.6,320,017). Other compounds with uncharged, hydrophilic, steric-barriermoieties, which prevent aggregation during formulation, like PEG, Gm1 orATTA, can also be coupled to lipids for use as in the methods andcompositions of the invention. ATTA-lipids are described, e.g., in U.S.Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., inU.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the present invention can have a varietyof “anchoring” lipid portions to secure the PEG portion to the surfaceof the lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mPEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications it maybe preferable for the nucleic acid-lipid particle to exhibit a longerplasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₀ toC₂₀ are preferred. In another group of embodiments, lipids with mono ordiunsaturated fatty acids with carbon chain lengths in the range of C₁₀to C₂₀ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the present invention are DOPE, DSPC, POPC, DPPC or anyrelated phosphatidylcholine. The neutral lipids useful in the presentinvention may also be composed of sphingomyelin, dihydrosphingomyeline,or phospholipids with other head groups, such as serine and inositol.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the present invention. Suchcationic lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3□-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

Anionic lipids suitable for use in lipid particles of the presentinvention include, but are not limited to, phosphatidylglycerol,cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,N-dodecanoyl phosphatidylethanoloamine, N-succinylphosphatidylethanolamine, N-glutaryl phosphatidylethanolamine,lysylphosphatidylglycerol, and other anionic modifying groups joined toneutral lipids.

In numerous embodiments, amphipathic lipids are included in lipidparticles of the present invention. “Amphipathic lipids” refer to anysuitable material, wherein the hydrophobic portion of the lipid materialorients into a hydrophobic phase, while the hydrophilic portion orientstoward the aqueous phase. Such compounds include, but are not limitedto, phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and □-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the presentinvention are programmable fusion lipids. Such lipid particles havelittle tendency to fuse with cell membranes and deliver their payloaduntil a given signal event occurs. This allows the lipid particle todistribute more evenly after injection into an organism or disease sitebefore it starts fusing with cells. The signal event can be, forexample, a change in pH, temperature, ionic environment, or time. In thelatter case, a fusion delaying or “cloaking” component, such as anATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange outof the lipid particle membrane over time. By the time the lipid particleis suitably distributed in the body, it has lost sufficient cloakingagent so as to be fusogenic. With other signal events, it is desirableto choose a signal that is associated with the disease site or targetcell, such as increased temperature at a site of inflammation.

In certain embodiments, it is desirable to target the lipid particles ofthis invention using targeting moieties that are specific to a cell typeor tissue. Targeting of lipid particles using a variety of targetingmoieties, such as ligands, cell surface receptors, glycoproteins,vitamins (e.g., riboflavin) and monoclonal antibodies, has beenpreviously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can comprise the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the target moiety is available for interaction withthe target, for example, a cell surface receptor. A variety of differenttargeting agents and methods are known and available in the art,including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In one exemplary embodiment, the lipid particle comprises a mixture of acationic lipid of the present invention, neutral lipids (other than acationic lipid), a sterol (e.g., cholesterol) and a PEG-modified lipid(e.g., a PEG-DMG or PEG-DMA). In certain embodiments, the lipid mixtureconsists of or consists essentially of a cationic lipid of the presentinvention, a neutral lipid, cholesterol, and a PEG-modified lipid. Infurther preferred embodiments, the lipid particle consists of orconsists essentially of the above lipid mixture in molar ratios of about20-70% amino lipid:5-45% neutral lipid:20-55% cholesterol:0.5-15%PEG-modified lipid.

In one embodiment, the lipid particle comprises at least two lipidsdisclosed herein. For example, a mixture of cationic lipids can be usedin a lipid particle, such that the mixture comprises 20-60% of the totallipid content on a molar basis.

In particular embodiments, the lipid particle consists of or consistsessentially of a cationic lipid chosen from Table 1, DSPC, Chol, andeither PEG-DMG or PEG-DMA, e.g., in a molar ratio of about 20-60%cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA. Inparticular embodiments, the molar lipid ratio is approximately40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA),35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). Inanother group of embodiments, the neutral lipid, DSPC, in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

Therapeutic Agent-Lipid Particle Compositions and Formulations

The present invention includes compositions comprising a lipid particleof the present invention and an active agent, wherein the active agentis associated with the lipid particle. In particular embodiments, theactive agent is a therapeutic agent. In particular embodiments, theactive agent is encapsulated within an aqueous interior of the lipidparticle. In other embodiments, the active agent is present within oneor more lipid layers of the lipid particle. In other embodiments, theactive agent is bound to the exterior or interior lipid surface of alipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade free nucleic acids. In afully encapsulated system, preferably less than 25% of particle nucleicacid is degraded in a treatment that would normally degrade 100% of freenucleic acid, more preferably less than 10% and most preferably lessthan 5% of the particle nucleic acid is degraded. Alternatively, fullencapsulation may be determined by an Oligreen® assay. Oligreen® is anultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. Therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide,levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

Nucleic Acid-Lipid Particles

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle. In particular embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” is meant to include any oligonucleotide or polynucleotide.Fragments containing up to 50 nucleotides are generally termedoligonucleotides, and longer fragments are called polynucleotides. Inparticular embodiments, oligonucleotides of the present invention are15-50 nucleotides in length.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA and other RNAinterference reagents. Single-stranded nucleic acids include, e.g.,antisense oligonucleotides, ribozymes, microRNA, and triplex-formingoligonucleotides. The nucleic acid that is present in a lipid-nucleicacid particle of this invention may include one or more of theoligonucleotide modifications described below.

Nucleic acids of the present invention may be of various lengths,generally dependent upon the particular form of nucleic acid. Forexample, in particular embodiments, plasmids or genes may be from about1,000 to 100,000 nucleotide residues in length. In particularembodiments, oligonucleotides may range from about 10 to 100 nucleotidesin length. In various related embodiments, oligonucleotides,single-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 50 nucleotides, from about 20 o about 50nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length.

In particular embodiments, the oligonucleotide (or a strand thereof) ofthe present invention specifically hybridizes to or is complementary toa target polynucleotide. “Specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility or expression therefrom, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions, e.g. mismatches, as compared to the region of a gene ormRNA sequence that it is targeting or to which it specificallyhybridizes.

RNA Interference Nucleic Acids

In particular embodiments, nucleic acid-lipid particles of the presentinvention are associated with RNA interference (RNAi) molecules. RNAinterference methods using RNAi molecules may be used to disrupt theexpression of a gene or polynucleotide of interest. Small interferingRNA (siRNA) has essentially replaced antisense ODN and ribozymes as thenext generation of targeted oligonucleotide drugs under development.

SiRNAs are RNA duplexes normally 16-30 nucleotides long that canassociate with a cytoplasmic multi-protein complex known as RNAi-inducedsilencing complex (RISC). RISC loaded with siRNA mediates thedegradation of homologous mRNA transcripts, therefore siRNA can bedesigned to knock down protein expression with high specificity. Unlikeother antisense technologies, siRNA function through a natural mechanismevolved to control gene expression through non-coding RNA. This isgenerally considered to be the reason why their activity is more potentin vitro and in vivo than either antisense ODN or ribozymes. A varietyof RNAi reagents, including siRNAs targeting clinically relevanttargets, are currently under pharmaceutical development, as described,e.g., in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

While the first described RNAi molecules were RNA:RNA hybrids comprisingboth an RNA sense and an RNA antisense strand, it has now beendemonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton, J. S. and Christian, A. T., (2003) Molecular Biotechnology24:111-119). Thus, the invention includes the use of RNAi moleculescomprising any of these different types of double-stranded molecules. Inaddition, it is understood that RNAi molecules may be used andintroduced to cells in a variety of forms. Accordingly, as used herein,RNAi molecules encompasses any and all molecules capable of inducing anRNAi response in cells, including, but not limited to, double-strandedoligonucleotides comprising two separate strands, i.e. a sense strandand an antisense strand, e.g., small interfering RNA (siRNA);double-stranded oligonucleotide comprising two separate strands that arelinked together by non-nucleotidyl linker; oligonucleotides comprising ahairpin loop of complementary sequences, which forms a double-strandedregion, e.g., shRNAi molecules, and expression vectors that express oneor more polynucleotides capable of forming a double-strandedpolynucleotide alone or in combination with another polynucleotide.

A “single strand siRNA compound” as used herein, is an siRNA compoundwhich is made up of a single molecule. It may include a duplexed region,formed by intra-strand pairing, e.g., it may be, or include, a hairpinor pan-handle structure. Single strand siRNA compounds may be antisensewith regard to the target molecule

A single strand siRNA compound may be sufficiently long that it canenter the RISC and participate in RISC mediated cleavage of a targetmRNA. A single strand siRNA compound is at least 14, and in otherembodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. In certain embodiments, it is less than 200, 100, or 60nucleotides in length.

Hairpin siRNA compounds will have a duplex region equal to or at least17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion will may be equal to or less than 200, 100, or 50, in length. Incertain embodiments, ranges for the duplex region are 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may havea single strand overhang or terminal unpaired region. In certainembodiments, the overhangs are 2-3 nucleotides in length. In someembodiments, the overhang is at the sense side of the hairpin and insome embodiments on the antisense side of the hairpin.

A “double stranded siRNA compound” as used herein, is an siRNA compoundwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded siRNA compound may be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. As used herein, term “antisense strand” means the strand of ansiRNA compound that is sufficiently complementary to a target molecule,e.g. a target RNA.

The sense strand of a double stranded siRNA compound may be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It may be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded siRNA compound may beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It may be equal to or less than200, 100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the siRNA compound is sufficiently large that itcan be cleaved by an endogenous molecule, e.g., by Dicer, to producesmaller siRNA compounds, e.g., siRNAs agents

The sense and antisense strands may be chosen such that thedouble-stranded siRNA compound includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-strandedsiRNA compound may contain sense and antisense strands, paired tocontain an overhang, e.g., one or two 5′ or 3′ overhangs, or a 3′overhang of 1-3 nucleotides. The overhangs can be the result of onestrand being longer than the other, or the result of two strands of thesame length being staggered. Some embodiments will have at least one 3′overhang. In one embodiment, both ends of an siRNA molecule will have a3′ overhang. In some embodiments, the overhang is 2 nucleotides.

In certain embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe ssiRNA compound range discussed above. ssiRNA compounds can resemblein length and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the ssiRNA compound arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also within the invention.

The siRNA compounds described herein, including double-stranded siRNAcompounds and single-stranded siRNA compounds can mediate silencing of atarget RNA, e.g., mRNA, e.g., a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an ssiRNA compound of 21 to23 nucleotides.

In one embodiment, an siRNA compound is “sufficiently complementary” toa target RNA, e.g., a target mRNA, such that the siRNA compound silencesproduction of protein encoded by the target mRNA. In another embodiment,the siRNA compound is “exactly complementary” to a target RNA, e.g., thetarget RNA and the siRNA compound anneal, for example to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in certain embodiments, thesiRNA compound specifically discriminates a single-nucleotidedifference. In this case, the siRNA compound only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are single stranded˜17-25 nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S.NAR, 2004, 32, Database Issue, D109-D111; and also athttp://microrna.sanger.ac.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence, e.g. a target gene mRNA.Antisense oligonucleotides are thought to inhibit gene expression bybinding to a complementary mRNA. Binding to the target mRNA can lead toinhibition of gene expression by through making the

either by preventing translation of complementary mRNA strands bybinding to it or by leading to degradation of the target mRNA AntisenseDNA can be used to target a specific, complementary (coding ornon-coding) RNA. If binding takes places this DNA/RNA hybrid can bedegraded by the enzyme RNase H. In particular embodiment, antisenseoligonucleotides contain from about 10 to about 50 nucleotides, morepreferably about 15 to about 30 nucleotides. The term also encompassesantisense oligonucleotides that may not be exactly complementary to thedesired target gene. Thus, the invention can be utilized in instanceswhere non-target specific-activities are found with antisense, or wherean antisense sequence containing one or more mismatches with the targetsequence is the most preferred for a particular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examplesof antisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Penis et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs by forming duplexes comprisingthe antagomir and endogenous miRNA, thereby preventing miRNA-inducedgene silencing. An example of antagomir-mediated miRNA silencing is thesilencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated by reference herein in itsentirety. Antagomir RNAs may be synthesized using standard solid phaseoligonucleotide synthesis protocols. See U.S. patent application Ser.Nos. 11/502,158 and 11/657,341 (the disclosure of each of which areincorporated herein by reference).

An antagomir can include ligand-conjugated monomer subunits and monomersfor oligonucleotide synthesis. Exemplary monomers are described in U.S.application Ser. No. 10/916,185, filed on Aug. 10, 2004. An antagomircan have a ZXY structure, such as is described in PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004. An antagomir can be complexedwith an amphipathic moiety. Exemplary amphipathic moieties for use witholigonucleotide agents are described in PCT Application No.PCT/US2004/07070, filed on Mar. 8, 2004.

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers may be RNA or DNA based, and may include a riboswitch.A riboswitch is a part of an mRNA molecule that can directly bind asmall target molecule, and whose binding of the target affects thegene's activity. Thus, an mRNA that contains a riboswitch is directlyinvolved in regulating its own activity, depending on the presence orabsence of its target molecule. Generally, aptamers are engineeredthrough repeated rounds of in vitro selection or equivalently, SELEX(systematic evolution of ligands by exponential enrichment) to bind tovarious molecular targets such as small molecules, proteins, nucleicacids, and even cells, tissues and organisms. The aptamer may beprepared by any known method, including synthetic, recombinant, andpurification methods, and may be used alone or in combination with otheraptamers specific for the same target. Further, as described more fullyherein, the term “aptamer” specifically includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA moleculescomplexes having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, JMol Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature.1992 May 14; 357(6374):173-6). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiet al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis □virus motif is described by Perrotta and Been, Biochemistry. 1992 Dec.1; 31(47):11843-52; an example of the RNaseP motif is described byGuerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the GroupI intron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used according tothe invention are that they have a specific substrate binding site whichis complementary to one or more of the target gene DNA or RNA regions,and that they have nucleotide sequences within or surrounding thatsubstrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal or other patient.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076),or CpG motifs, as well as other known ISS features (such as multi-Gdomains, see WO 96/11266).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” According to the present invention, such an oligonucleotide isconsidered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In a specific embodiment, thenucleic acid comprises the sequence 5′ TAACGTTGAGGGGCAT 3′. In analternative embodiment, the nucleic acid comprises at least two CpGdinucleotides, wherein at least one cytosine in the CpG dinucleotides ismethylated. In a further embodiment, each cytosine in the CpGdinucleotides present in the sequence is methylated. In anotherembodiment, the nucleic acid comprises a plurality of CpG dinucleotides,wherein at least one of said CpG dinucleotides comprises a methylatedcytosine.

In one specific embodiment, the nucleic acid comprises the sequence 5′TTCCATGACGTTCCTGACGT 3′. In another specific embodiment, the nucleicacid sequence comprises the sequence 5′ TCCATGACGTTCCTGACGT 3′, whereinthe two cytosines indicated in bold are methylated. In particularembodiments, the ODN is selected from a group of ODNs consisting of ODN#1, ODN #2, ODN #3, ODN #4, ODN #5, ODN #6, ODN #7, ODN #8, and ODN #9,as shown below.

TABLE 3 Exemplary Immunostimulatory Oligonucleotides (ODNs) SEQ ODN NAMEID ODN SEQUENCE (5′-3′). ODN 1 NO: 1  5′-TAACGTTGAGGGGCAT-3 human c-myc* ODN 1m NO: 2  5′-TAAZGTTGAGGGGCAT-3 ODN 2 NO: 3 5′-TCCATGACGTTCCTGACGTT-3 * ODN 2m NO: 4  5′-TCCATGAZGTTCCTGAZGTT-3ODN 3 NO: 5  5′-TAAGCATACGGGGTGT-3 ODN 5 5′-AACGTT-3 ODN 6 NO: 6 5′-GATGCTGTGTCGGGGTCTCCGGGC-3′ ODN 7 NO: 7 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ ODN 7m NO: 8 5′-TZGTZGTTTTGTZGTTTTGTZGTT-3′ ODN 8 NO: 9  5′-TCCAGGACTTCTCTCAGGTT-3′ODN 9 NO: 10 5′-TCTCCCAGCGTGCGCCAT-3′ ODN 10 murine NO: 115′-TGCATCCCCCAGGCCACCAT-3 Intracellular Adhesion Molecule-1 ODN 11 humanNO: 12 5′-GCCCAAGCTGGCATCCGTCA-3′ Intracellular Adhesion Molecule-1ODN 12 human NO: 13 5′-GCCCAAGCTGGCATCCGTCA-3′ IntracellularAdhesion Molecule-1 ODN 13 human erb-B-2 NO: 14 5′-GGT GCTCACTGC GGC-3′ODN 14 human c-myc NO: 15 5′-AACC GTT GAG GGG CAT-3′ ODN 15 human c-mycNO: 16 5′-TAT GCT GTG CCG GGG TCT TCG GGC-3′ ODN 16 NO: 175′-GTGCCG GGGTCTTCGGGC-3′ ODN 17 human Insulin NO: 185′-GGACCCTCCTCCGGAGCC-3′ Growth Factor 1- Receptor ODN 18 human InsulinNO: 19 5′-TCC TCC GGA GCC AGA CTT-3′ Growth Factor 1- ReceptorODN 19 human Epidermal NO: 20 5′-AAC GTT GAG GGG CAT-3′Growth Factor- Receptor ODN 20 Epidermal Growth NO: 215′-CCGTGGTCA TGCTCC-3′ Factor- Receptor ODN 21 human Vascular NO: 225′-CAG CCTGGCTCACCG CCTTGG-3′ Endothelial Growth Factor ODN 22 murineNO: 23 5′-CAG CCA TGG TTC CCC CCA AC-3′ Phosphokinase C- alpha ODN 23NO: 24 5′-GTT CTC GCT GGT GAG TTT CA-3′ ODN 24 human Bcl-2 NO: 255′-TCT CCCAGCGTGCGCCAT-3′ ODN 25 human C-Raf-s NO: 265′-GTG CTC CAT TGA TGC-3′ ODN #26 human Vascular NO: 275′-GAGUUCUGAUGAGGCCGAAAGG Endothelial Growth CCGAAAGUCUG-3′Factor Receptor-1 ODN #27 5′-RRCGYY-3′ ODN #28 NO: 285′-AACGTTGAGGGGCAT-3′ ODN #29 NO: 29 5′-CAACGTTATGGGGAGA-3′ODN #30 human c-myc NO: 30 5′-TAACGTTGAGGGGCAT-3′ “Z” represents amethylated cytosine residue. ODN 14 is a 15-mer oligonucleotide and ODN1 is the same oligonucleotide having a thymidine added onto the 5′ endmaking ODN 1 into a 16-mer. No difference in biological activity betweenODN 14 and ODN 1 has been detected and both exhibit similarimmunostimulatory activity (Mui et al., 2001)

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in Raney et al., Journal of Pharmacology and ExperimentalTherapeutics, 298:1185-1192 (2001). In certain embodiments, ODNs used inthe compositions and methods of the present invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Decoy Oligonucleotides

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety

Supermir

A supermir refers to a single stranded, double stranded or partiallydouble stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or both or modifications thereof, which hasa nucleotide sequence that is substantially identical to an miRNA andthat is antisense with respect to its target. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages and which contain at leastone non-naturally-occurring portion which functions similarly. Suchmodified or substituted oligonucleotides are preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. In a preferred embodiment, thesupermir does not include a sense strand, and in another preferredembodiment, the supermir does not self-hybridize to a significantextent. An supermir featured in the invention can have secondarystructure, but it is substantially single-stranded under physiologicalconditions. An supermir that is substantially single-stranded issingle-stranded to the extent that less than about 50% (e.g., less thanabout 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed withitself. The supermir can include a hairpin segment, e.g., sequence,preferably at the 3′ end can self hybridize and form a duplex region,e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than8, 7, 6, or n nucleotides, e.g., 5 nucleotides. The duplexed region canbe connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6dTs, e.g., modified dTs. In another embodiment the supermir is duplexedwith a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10 nucleotides inlength, e.g., at one or both of the 3′ and 5′ end or at one end and inthe non-terminal or middle of the supermir.

miRNA Mimics

miRNA mimics represent a class of molecules that can be used to imitatethe gene silencing ability of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs). miRNA mimics can becomprised of nucleic acid (modified or modified nucleic acids) includingoligonucleotides comprising, without limitation, RNA, modified RNA, DNA,modified DNA, locked nucleic acids, or 2′-O,4′-C-ethylene-bridgednucleic acids (ENA), or any combination of the above (including DNA-RNAhybrids). In addition, miRNA mimics can comprise conjugates that canaffect delivery, intracellular compartmentalization, stability,specificity, functionality, strand usage, and/or potency. In one design,miRNA mimics are double stranded molecules (e.g., with a duplex regionof between about 16 and about 31 nucleotides in length) and contain oneor more sequences that have identity with the mature strand of a givenmiRNA. Modifications can comprise 2′ modifications (including 2′-Omethyl modifications and 2′ F modifications) on one or both strands ofthe molecule and internucleotide modifications (e.g. phorphorthioatemodifications) that enhance nucleic acid stability and/or specificity.In addition, miRNA mimics can include overhangs. The overhangs canconsist of 1-6 nucleotides on either the 3′ or 5′ end of either strandand can be modified to enhance stability or functionality. In oneembodiment, a miRNA mimic comprises a duplex region of between 16 and 31nucleotides and one or more of the following chemical modificationpatterns: the sense strand contains 2′-O-methyl modifications ofnucleotides 1 and 2 (counting from the 5′ end of the senseoligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise 2′ F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Antimir or miRNA Inhibitor.

The terms “antimir” “microRNA inhibitor”, “miR inhibitor”, or“inhibitor” are synonymous and refer to oligonucleotides or modifiedoligonucleotides that interfere with the ability of specific miRNAs. Ingeneral, the inhibitors are nucleic acid or modified nucleic acids innature including oligonucleotides comprising RNA, modified RNA, DNA,modified DNA, locked nucleic acids (LNAs), or any combination of theabove. Modifications include 2′ modifications (including 2′-0 alkylmodifications and 2′ F modifications) and internucleotide modifications(e.g. phosphorothioate modifications) that can affect delivery,stability, specificity, intracellular compartmentalization, or potency.In addition, miRNA inhibitors can comprise conjugates that can affectdelivery, intracellular compartmentalization, stability, and/or potency.Inhibitors can adopt a variety of configurations including singlestranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpindesigns, in general, microRNA inhibitors comprise contain one or moresequences or portions of sequences that are complementary or partiallycomplementary with the mature strand (or strands) of the miRNA to betargeted, in addition, the miRNA inhibitor may also comprise additionalsequences located 5′ and 3′ to the sequence that is the reversecomplement of the mature miRNA. The additional sequences may be thereverse complements of the sequences that are adjacent to the maturemiRNA in the pri-miRNA from which the mature miRNA is derived, or theadditional sequences may be arbitrary sequences (having a mixture of A,G, C, or U). In some embodiments, one or both of the additionalsequences are arbitrary sequences capable of forming hairpins. Thus, insome embodiments, the sequence that is the reverse complement of themiRNA is flanked on the 5′ side and on the 3′ side by hairpinstructures. Micro-RNA inhibitors, when double stranded, may includemismatches between nucleotides on opposite strands. Furthermore,micro-RNA inhibitors may be linked to conjugate moieties in order tofacilitate uptake of the inhibitor into a cell. For example, a micro-RNAinhibitor may be linked to cholesteryl5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate) whichallows passive uptake of a micro-RNA inhibitor into a cell. Micro-RNAinhibitors, including hairpin miRNA inhibitors, are described in detailin Vermeulen et al., “Double-Stranded Regions Are Essential DesignComponents Of Potent Inhibitors of RISC Function,” RNA 13: 723-730(2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

U1 Adaptor

U1 adaptor inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘U1 domain’ that binds to the U1 smaller nuclear RNAcomponent of the U1 snRNP (Goraczniak, et al., 2008, NatureBiotechnology, 27(3), 257-263, which is expressly incorporated byreference herein, in its entirety). U1 snRNP is a ribonucleoproteincomplex that functions primarily to direct early steps in spliceosomeformation by binding to the pre-mRNA exon-intron boundary (Brown andSimpson, 1998, Annu Rev Plant Physiol Plant MoI Biol 49:77-95).Nucleotides 2-11 of the 5′end of U1 snRNA base pair bind with the 5′ssof the pre mRNA. In one embodiment, oligonucleotides of the inventionare U1 adaptors. In one embodiment, the U1 adaptor can be administeredin combination with at least one other iRNA agent.

Oligonucleotide Modifications

Unmodified oligonucleotides may be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications ofoligonucleotides can confer improved properties, and, e.g., can renderoligonucleotides more stable to nucleases.

As oligonucleotides are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin an oligonucleotide, e.g., a modification of a base, a sugar, aphosphate moiety, or the non-bridging oxygen of a phosphate moiety. Itis not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even ata single nucleoside within an oligonucleotide.

In some cases the modification will occur at all of the subjectpositions in the oligonucleotide but in many, and in fact in most casesit will not. By way of example, a modification may only occur at a 3′ or5′ terminal position, may only occur in the internal region, may onlyoccur in a terminal regions, e.g. at a position on a terminal nucleotideor in the last 2, 3, 4, 5, or 10 nucleotides of an oligonucleotide. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of a double-stranded oligonucleotide or may only occur in asingle strand region of a double-stranded oligonucleotide. E.g., aphosphorothioate modification at a non-bridging oxygen position may onlyoccur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

A modification described herein may be the sole modification, or thesole type of modification included on multiple nucleotides, or amodification can be combined with one or more other modificationsdescribed herein. The modifications described herein can also becombined onto an oligonucleotide, e.g. different nucleotides of anoligonucleotide have different modifications described herein.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular nucleobases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. In certain embodiments, one of the non-bridgingphosphate oxygen atoms in the phosphate backbone moiety can be replacedby any of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl), C(i.e. an alkyl group, an aryl group, etc. . . . ), H, NR₂ (R ishydrogen, alkyl, aryl), or OR (R is alkyl or aryl). The phosphorous atomin an unmodified phosphate group is achiral. However, replacement of oneof the non-bridging oxygens with one of the above atoms or groups ofatoms renders the phosphorous atom chiral; in other words a phosphorousatom in a phosphate group modified in this way is a stereogenic center.The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotides diastereomers. Thus,while not wishing to be bound by theory, modifications to bothnon-bridging oxygens, which eliminate the chiral center, e.g.phosphorodithioate formation, may be desirable in that they cannotproduce diastereomer mixtures. Thus, the non-bridging oxygens can beindependently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includemethyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include themethylenecarbonylamino and methylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Sugar Modifications

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an oligonucleotide can include nucleotidescontaining e.g., arabinose, as the sugar. The monomer can have an alphalinkage at the 1′ position on the sugar, e.g., alpha-nucleosides.Oligonucleotides can also include “abasic” sugars, which lack anucleobase at C-1′. These abasic sugars can also be further containingmodifications at one or more of the constituent sugar atoms.Oligonucleotides can also contain one or more sugars that are in the Lform, e.g. L-nucleosides.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a linker. The terminal atom of the linker canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the linkercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs).

When a linker/phosphate-functional molecular entity-linker/phosphatearray is interposed between two strands of a dsRNA, this array cansubstitute for a hairpin RNA loop in a hairpin-type RNA agent.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments antisense strands of dsRNAs, are 5′ phosphorylatedor include a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)2(S)P—O—5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natural bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine,N-methylguanines, or O-alkylated bases. Further purines and pyrimidinesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inthe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and thosedisclosed by Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613.

Cationic Groups

Modifications to oligonucleotides can also include attachment of one ormore cationic groups to the sugar, base, and/or the phosphorus atom of aphosphate or modified phosphate backbone moiety. A cationic group can beattached to any atom capable of substitution on a natural, unusual oruniversal base. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, ordiheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Placement within an Oligonucleotide

Some modifications may preferably be included on an oligonucleotide at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of an oligonucleotide. A preferred location of amodification on an oligonucleotide, may confer preferred properties onthe agent. For example, preferred locations of particular modificationsmay confer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity.

One or more nucleotides of an oligonucleotide may have a 2′-5′ linkage.One or more nucleotides of an oligonucleotide may have invertedlinkages, e.g. 3′-3′, 5′-5′, 2′-2′ or 2′-3′ linkages.

A double-stranded oligonucleotide may include at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide, or a terminal 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide, or aterminal 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide, or a terminal 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-uridine-3′ (5′-CU-3′)dinucleotide, wherein the 5% cytidine is a 2′-modified nucleotide, or aterminal 5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide. Double-stranded oligonucleotidesincluding these modifications are particularly stabilized againstendonuclease activity.

General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter 2, Oligoribonucleotide synthesis, Chapter 3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4,Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7, 651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Nucleobases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In one embodiment, the linker is—[(P-Q-R)_(q)—X—(P′-Q′-R′)_(q′)]_(q″)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q and Q′ are each independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹)(R²)(CH₂)_(n)—, —(CH₂)—C(R¹)(R²)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum). Cleavable linkinggroups are susceptible to cleavage agents, e.g., pH, redox potential orthe presence of degradative molecules. Generally, cleavage agents aremore prevalent or found at higher levels or activities inside cells thanin serum or blood. Examples of such degradative agents include: redoxagents which are selected for particular substrates or which have nosubstrate specificity, including, e.g., oxidative or reductive enzymesor reductive agents such as mercaptans, present in cells, that candegrade a redox cleavable linking group by reduction; esterases;endosomes or agents that can create an acidic environment, e.g., thosethat result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable linking group by acting as a general acid,peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Ligands

A wide variety of entities can be coupled to the oligonucleotides andlipids of the present invention. Preferred moieties are ligands, whichare coupled, preferably covalently, either directly or indirectly via anintervening tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Ligands providing enhancedaffinity for a selected target are also termed targeting ligands.Preferred ligands for conjugation to the lipids of the present inventionare targeting ligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in Table 4.

TABLE 4 List of peptides with endosomolytic activity. Name SEQ IDSequence (N to C) Ref. GALA NO: 31 AALEALAEALEALAEALEALAEAAAA 1 GGC EALANO: 32 AALAEALAEALAEALAEALAEALAAA 2 AGGC NO: 33 ALEALAEALEALAEA 3 INF-7NO: 34 GLFEAIEGFIENGWEGMIWDYG 4 Inf HA-2 NO: 35 GLFGAIAGFIENGWEGMIDGWYG5 diINF-7 NO: 36 GLF EAI EGFI ENGW EGMI DGWYGC 5GLF EAI EGFI ENGW EGMI DGWYGC diINF3 NO: 37 GLF EAI EGFI ENGW EGMI DGGC6 GLF EAI EGFI ENGW EGMI DGGC GLF NO: 38 GLFGALAEALAEALAEHLAEALAEALE 6ALAAGGSC GALA-INF3 NO: 39 GLFEAIEGFIENGWEGLAEALAEALEA 6 LAAGGSC INF-5NO: 40 GLF EAI EGFI ENGW EGnI DG K 4 NO: 41 GLF EAI EGFI ENGW EGnI DG n,norleucine References 1. Subbarao et al., Biochemistry, 1987, 26:2964-2972. 2. Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586 3.Turk, M. J., Reddy, J. A. et al. (2002). Characterization of a novelpH-sensitive peptide that enhances drug release from folate-targetedliposomes at endosomal pHs. Biochim. Biophys. Acta 1559, 56-68. 4.Plank, C. Oberhauser, B. Mechtler, K. Koch, C. Wagner, E. (1994). Theinfluence of endosome-disruptive peptides on gene transfer usingsynthetic virus-like gene transfer systems, J. Biol. Chem. 26912918-12924. 5. Mastrobattista, E., Koning, G. A. et al. (2002).Functional characterization of an endosome-disruptive peptide and itsapplication in cytosolic delivery of immunoliposome-entrapped proteins.J. Biol. Chem. 277, 27135-43. 6. Oberhauser, B., Plank, C. et al.(1995). Enhancing endosomal exit of nucleic acids using pH-sensitiveviral fusion peptides. Deliv. Strategies Antisense Oligonucleotide Ther.247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 5 shows some examples of targetingligands and their associated receptors.

TABLE 5 Targeting Ligands and their associated receptors Liver CellsLigand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial Cell ProcollagenProcollagen receptor (SEC) Negatively charged molecules Scavengerreceptors Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell Mannose Mannose receptors(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40 K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long (see Table 6, for example).

TABLE 6 Exemplary Cell Permeation Peptides. Cell Permeation PeptideSEQ ID Amino acid Sequence Reference Penetratin NO:42 RQIKIWFQNRRMKWKKDerossi et al., J. Biol. Chem. 269:10444, 1994 Tat fragment NO:43GRKKRRQRRRPPQC Vives et al., J. Biol. (48-60) Chem., 272:16010, 1997Signal Sequence- NO:44 GALFLGWLGAAGSTMGAWS Chaloin et al., Biochem.based peptide QPKKKRKV Biophys. Res. Commun., 243:601, 1998 PVEC NO:45LLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell Res., 269:237, 2001Transportan NO:46 GWTLNSAGYLLKINLKALA Pooga et al., FASEB J., ALAKKIL12:67, 1998 Amphiphilic model NO:47 KLALKLALKALKAALKLAOehlke et al., Mol. Ther., peptide 2:339, 2000 Arg₉ NO:48 RRRRRRRRRMitchell et al., J. Pept. Res., 56:318, 2000 Bacterial cell NO:49KFFKFFKFFK wall permeating LL-37 NO:50 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Cecropin P1 NO:51 SWLSKTAKKLENSAKKRIS EGIAIAIQGGPRα-defensin NO:52 ACYCRIPACIAGERRYGTC IYQGRLWAFCC b-defensin NO:53DHYNCVSSGGQCLYSACPI FTKIQGTCYRGKAKCCK Bactenecin NO:54 RKCRIVVIRVCRPR-39 NO:55 RRRPRPPYLPRPRPPPFFP PRLPPRIPPGFPPRFPPRF PGKR-NH2 IndolicidinNO:56 ILPWKWPWWPWRR-NH2

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:57). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:58)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:59)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:60))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an αvβ3 integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the αvβ3 integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis. Preferred conjugates of this type ligands that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic monomeric units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether. The ligand or tethered ligand may be present on amonomer when said monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated via coupling to a“precursor” monomer after said “precursor” monomer has been incorporatedinto the growing strand. For example, a monomer having, e.g., anamino-terminated tether (i.e., having no associated ligand), e.g.,TAP-(CH₂)_(n)—NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer into the strand, a ligand having an electrophilicgroup, e.g., a pentafluorophenyl ester or aldehyde group, cansubsequently be attached to the precursor monomer by coupling theelectrophilic group of the ligand with the terminal nucleophilic groupof the precursor monomer's tether.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662; 5,688,941;5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437;6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559,279; eachof which is herein incorporated by reference.

Characteristic of Nucleic Acid-Lipid Particles

In certain embodiments, the present invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, incorporating siRNA oligonucleotides, arecharacterized using a variety of biophysical parameters including: (1)drug to lipid ratio; (2) encapsulation efficiency; and (3) particlesize. High drug to lipid rations, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter are desirable. In addition, thenature of the nucleic acid polymer is of significance, since themodification of nucleic acids in an effort to impart nuclease resistanceadds to the cost of therapeutics while in many cases providing onlylimited resistance. Unless stated otherwise, these criteria arecalculated in this specification as follows:

Nucleic acid to lipid ratio is the amount of nucleic acid in a definedvolume of preparation divided by the amount of lipid in the same volume.This may be on a mole per mole basis or on a weight per weight basis, oron a weight per mole basis. For final, administration-readyformulations, the nucleic acid:lipid ratio is calculated after dialysis,chromatography and/or enzyme (e.g., nuclease) digestion has beenemployed to remove as much of the external nucleic acid as possible;

Encapsulation efficiency refers to the drug to lipid ratio of thestarting mixture divided by the drug to lipid ratio of the final,administration competent formulation. This is a measure of relativeefficiency. For a measure of absolute efficiency, the total amount ofnucleic acid added to the starting mixture that ends up in theadministration competent formulation, can also be calculated. The amountof lipid lost during the formulation process may also be calculated.Efficiency is a measure of the wastage and expense of the formulation;and

Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer. Particles under200 nm are preferred for distribution to neo-vascularized (leaky)tissues, such as neoplasms and sites of inflammation.

Pharmaceutical Compositions

The lipid particles of present invention, particularly when associatedwith a therapeutic agent, may be formulated as a pharmaceuticalcomposition, e.g., which further comprises a pharmaceutically acceptablediluent, excipient, or carrier, such as physiological saline orphosphate buffer, selected in accordance with the route ofadministration and standard pharmaceutical practice.

In particular embodiments, pharmaceutical compositions comprising thelipid-nucleic acid particles of the invention are prepared according tostandard techniques and further comprise a pharmaceutically acceptablecarrier. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. In compositions comprising saline or othersalt containing carriers, the carrier is preferably added followinglipid particle formation. Thus, after the lipid-nucleic acidcompositions are formed, the compositions can be diluted intopharmaceutically acceptable carriers such as normal saline.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.01%, usually at or at least about 0.05-5% to as much as 10 to30% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. For example, the concentration may be increasedto lower the fluid load associated with treatment. This may beparticularly desirable in patients having atherosclerosis-associatedcongestive heart failure or severe hypertension. Alternatively,complexes composed of irritating lipids may be diluted to lowconcentrations to lessen inflammation at the site of administration. Inone group of embodiments, the nucleic acid will have an attached labeland will be used for diagnosis (by indicating the presence ofcomplementary nucleic acid). In this instance, the amount of complexesadministered will depend upon the particular label used, the diseasestate being diagnosed and the judgement of the clinician but willgenerally be between about 0.01 and about 50 mg per kilogram of bodyweight, preferably between about 0.1 and about 5 mg/kg of body weight.

As noted above, the lipid-therapeutic agent (e.g., nucleic acid)particles of the invention may include polyethylene glycol(PEG)-modified phospholipids, PEG-ceramide, or gangliosideG_(M1)-modified lipids or other lipids effective to prevent or limitaggregation. Addition of such components does not merely prevent complexaggregation. Rather, it may also provide a means for increasingcirculation lifetime and increasing the delivery of the lipid-nucleicacid composition to the target tissues.

The present invention also provides lipid-therapeutic agent compositionsin kit form. The kit will typically be comprised of a container that iscompartmentalized for holding the various elements of the kit. The kitwill contain the particles or pharmaceutical compositions of the presentinvention, preferably in dehydrated or concentrated form, withinstructions for their rehydration or dilution and administration. Incertain embodiments, the particles comprise the active agent, while inother embodiments, they do not.

Methods of Manufacture

The methods and compositions of the invention make use of certaincationic lipids, the synthesis, preparation and characterization ofwhich is described below and in the accompanying Examples. In addition,the present invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. In the methods described herein, a mixture of lipids is combinedwith a buffered aqueous solution of nucleic acid to produce anintermediate mixture containing nucleic acid encapsulated in lipidparticles wherein the encapsulated nucleic acids are present in anucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5to 15 wt %. The intermediate mixture may optionally be sized to obtainlipid-encapsulated nucleic acid particles wherein the lipid portions areunilamellar vesicles, preferably having a diameter of 30 to 150 nm, morepreferably about 40 to 90 nm. The pH is then raised to neutralize atleast a portion of the surface charges on the lipid-nucleic acidparticles, thus providing an at least partially surface-neutralizedlipid-encapsulated nucleic acid composition.

As described above, several of these cationic lipids are amino lipidsthat are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These cationic lipidsare termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Particularly advantageous aspects of this process includeboth the facile removal of any surface adsorbed nucleic acid and aresultant nucleic acid delivery vehicle which has a neutral surface.Liposomes or lipid particles having a neutral surface are expected toavoid rapid clearance from circulation and to avoid certain toxicitieswhich are associated with cationic liposome preparations. Additionaldetails concerning these uses of such titratable cationic lipids in theformulation of nucleic acid-lipid particles are provided in U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein byreference.

It is further noted that the vesicles formed in this manner provideformulations of uniform vesicle size with high content of nucleic acids.Additionally, the vesicles have a size range of from about 30 to about150 nm, more preferably about 30 to about 90 nm.

Without intending to be bound by any particular theory, it is believedthat the very high efficiency of nucleic acid encapsulation is a resultof electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) thevesicle surface is charged and binds a portion of the nucleic acidsthrough electrostatic interactions. When the external acidic buffer isexchanged for a more neutral buffer (e.g., pH 7.5) the surface of thelipid particle or liposome is neutralized, allowing any external nucleicacid to be removed. More detailed information on the formulation processis provided in various publications (e.g., U.S. Pat. No. 6,287,591 andU.S. Pat. No. 6,858,225).

In view of the above, the present invention provides methods ofpreparing lipid/nucleic acid formulations. In the methods describedherein, a mixture of lipids is combined with a buffered aqueous solutionof nucleic acid to produce an intermediate mixture containing nucleicacid encapsulated in lipid particles, e.g., wherein the encapsulatednucleic acids are present in a nucleic acid/lipid ratio of about 10 wt %to about 20 wt %. The intermediate mixture may optionally be sized toobtain lipid-encapsulated nucleic acid particles wherein the lipidportions are unilamellar vesicles, preferably having a diameter of 30 to150 nm, more preferably about 40 to 90 nm. The pH is then raised toneutralize at least a portion of the surface charges on thelipid-nucleic acid particles, thus providing an at least partiallysurface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first lipid component of the present invention thatis selected from among lipids which have a pKa such that the lipid iscationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel cationic lipid of thepresent invention.

In preparing the nucleic acid-lipid particles of the invention, themixture of lipids is typically a solution of lipids in an organicsolvent. This mixture of lipids can then be dried to form a thin film orlyophilized to form a powder before being hydrated with an aqueousbuffer to form liposomes. Alternatively, in a preferred method, thelipid mixture can be solubilized in a water miscible alcohol, such asethanol, and this ethanolic solution added to an aqueous bufferresulting in spontaneous liposome formation. In most embodiments, thealcohol is used in the form in which it is commercially available. Forexample, ethanol can be used as absolute ethanol (100%), or as 95%ethanol, the remainder being water. This method is described in moredetail in U.S. Pat. No. 5,976,567).

In one exemplary embodiment, the mixture of lipids is a mixture ofcationic lipids, neutral lipids (other than a cationic lipid), a sterol(e.g., cholesterol) and a PEG-modified lipid (e.g., a PEG-DMG orPEG-DMA) in an alcohol solvent. In preferred embodiments, the lipidmixture consists essentially of a cationic lipid, a neutral lipid,cholesterol and a PEG-modified lipid in alcohol, more preferablyethanol. In further preferred embodiments, the first solution consistsof the above lipid mixture in molar ratios of about 20-70% cationiclipid: 5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modifiedlipid. In still further preferred embodiments, the first solutionconsists essentially of a lipid chosen from Table 1, DSPC, Chol andPEG-DMG or PEG-DMA, more preferably in a molar ratio of about 20-60%cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA. Inparticular embodiments, the molar lipid ratio is approximately40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA),35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). Inanother group of preferred embodiments, the neutral lipid in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

In accordance with the invention, the lipid mixture is combined with abuffered aqueous solution that may contain the nucleic acids. Thebuffered aqueous solution of is typically a solution in which the bufferhas a pH of less than the pK_(a) of the protonatable lipid in the lipidmixture. Examples of suitable buffers include citrate, phosphate,acetate, and MES. A particularly preferred buffer is citrate buffer.Preferred buffers will be in the range of 1-1000 mM of the anion,depending on the chemistry of the nucleic acid being encapsulated, andoptimization of buffer concentration may be significant to achievinghigh loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.No. 6,858,225). Alternatively, pure water acidified to pH 5-6 withchloride, sulfate or the like may be useful. In this case, it may besuitable to add 5% glucose, or another non-ionic solute which willbalance the osmotic potential across the particle membrane when theparticles are dialyzed to remove ethanol, increase the pH, or mixed witha pharmaceutically acceptable carrier such as normal saline. The amountof nucleic acid in buffer can vary, but will typically be from about0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL toabout 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a)of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions is adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (HBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid. As describedabove, the aqueous buffer should be of a pH below the pKa of the aminolipid. A solution of the nucleic acids can then be added to these sized,preformed vesicles. To allow encapsulation of nucleic acids into such“pre-formed” vesicles the mixture should contain an alcohol, such asethanol. In the case of ethanol, it should be present at a concentrationof about 20% (w/w) to about 45% (w/w). In addition, it may be necessaryto warm the mixture of pre-formed vesicles and nucleic acid in theaqueous buffer-ethanol mixture to a temperature of about 25° C. to about50° C. depending on the composition of the lipid vesicles and the natureof the nucleic acid. It will be apparent to one of ordinary skill in theart that optimization of the encapsulation process to achieve a desiredlevel of nucleic acid in the lipid vesicles will require manipulation ofvariable such as ethanol concentration and temperature. Examples ofsuitable conditions for nucleic acid encapsulation are provided in theExamples. Once the nucleic acids are encapsulated within the prefromedvesicles, the external pH can be increased to at least partiallyneutralize the surface charge. Unencapsulated and surface adsorbednucleic acids can then be removed as described above.

Method of Use

The lipid particles of the present invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using a nucleic acid-lipid particles of the present invention.While the following description o various methods of using the lipidparticles and related pharmaceutical compositions of the presentinvention are exemplified by description related to nucleic acid-lipidparticles, it is understood that these methods and compositions may bereadily adapted for the delivery of any therapeutic agent for thetreatment of any disease or disorder that would benefit from suchtreatment.

In certain embodiments, the present invention provides methods forintroducing a nucleic acid into a cell. Preferred nucleic acids forintroduction into cells are siRNA, immune-stimulating oligonucleotides,plasmids, antisense and ribozymes. These methods may be carried out bycontacting the particles or compositions of the present invention withthe cells for a period of time sufficient for intracellular delivery tooccur.

The compositions of the present invention can be adsorbed to almost anycell type. Once adsorbed, the nucleic acid-lipid particles can either beendocytosed by a portion of the cells, exchange lipids with cellmembranes, or fuse with the cells. Transfer or incorporation of thenucleic acid portion of the complex can take place via any one of thesepathways. Without intending to be limited with respect to the scope ofthe invention, it is believed that in the case of particles taken upinto the cell by endocytosis the particles then interact with theendosomal membrane, resulting in destabilization of the endosomalmembrane, possibly by the formation of non-bilayer phases, resulting inintroduction of the encapsulated nucleic acid into the cell cytoplasm.Similarly in the case of direct fusion of the particles with the cellplasma membrane, when fusion takes place, the liposome membrane isintegrated into the cell membrane and the contents of the liposomecombine with the intracellular fluid. Contact between the cells and thelipid-nucleic acid compositions, when carried out in vitro, will takeplace in a biologically compatible medium. The concentration ofcompositions can vary widely depending on the particular application,but is generally between about 1 μmol and about 10 mmol. In certainembodiments, treatment of the cells with the lipid-nucleic acidcompositions will generally be carried out at physiological temperatures(about 37° C.) for periods of time from about 1 to 24 hours, preferablyfrom about 2 to 8 hours. For in vitro applications, the delivery ofnucleic acids can be to any cell grown in culture, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of embodiments, a lipid-nucleic acid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/mL, more preferably about 2×10⁴ cells/mL.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

In another embodiment, the lipid particles of the invention can be maybe used to deliver a nucleic acid to a cell or cell line (for example, atumor cell line). Non-limiting examples of such cell lines include: HELA(ATCC Cat N: CCL-2), KB (ATCC Cat N: CCL-17), HEP3B (ATCC Cat N:HB-8064), SKOV-3 (ATCC Cat N: HTB-77), HCT-116 (ATCC Cat N: CCL-247),HT-29 (ATCC Cat N: HTB-38), PC-3 (ATCC Cat N: CRL-1435), A549 (ATCC CatN: CCL-185), MDA-MB-231 (ATCC Cat N: HTB-26).

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products (i.e., for Duchenne's dystrophy, seeKunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cysticfibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for thecompositions of the present invention include introduction of antisenseoligonucleotides in cells (see, Bennett, et al., Mol. Pharm.41:1023-1033 (1992)).

Alternatively, the compositions of the present invention can also beused for deliver of nucleic acids to cells in vivo, using methods whichare known to those of skill in the art. With respect to delivery of DNAor mRNA sequences, Zhu, et al., Science 261:209-211 (1993), incorporatedherein by reference, describes the intravenous delivery ofcytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expressionplasmid using DOTMA-DOPE complexes. Hyde, et al., Nature 362:250-256(1993), incorporated herein by reference, describes the delivery of thecystic fibrosis transmembrane conductance regulator (CFTR) gene toepithelia of the airway and to alveoli in the lung of mice, usingliposomes. Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989),incorporated herein by reference, describes the in vivo transfection oflungs of mice with a functioning prokaryotic gene encoding theintracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, thecompositions of the invention can be used in the treatment of infectiousdiseases.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly. Inparticular embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For oneexample, see Stadler, et al., U.S. Pat. No. 5,286,634, which isincorporated herein by reference. Intracellular nucleic acid deliveryhas also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY,Academic Press, New York. 101:512-527 (1983); Mannino, et al.,Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278(1993). Still other methods of administering lipid-based therapeuticsare described in, for example, Rahman et al., U.S. Pat. No. 3,993,754;Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No.4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat.No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical,” it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The lipid-nucleic acid compositions can also be administered in anaerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci.298(4):278-281 (1989)) or by direct injection at the site of disease(Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, NewYork. pp. 70-71 (1994)).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

Dosages for the lipid-therapeutic agent particles of the presentinvention will depend on the ratio of therapeutic agent to lipid and theadministrating physician's opinion based on age, weight, and conditionof the patient.

In one embodiment, the present invention provides a method of modulatingthe expression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of thepresent invention that is associated with a nucleic acid capable ofmodulating the expression of a target polynucleotide or polypeptide. Asused herein, the term “modulating” refers to altering the expression ofa target polynucleotide or polypeptide. In different embodiments,modulating can mean increasing or enhancing, or it can mean decreasingor reducing. Methods of measuring the level of expression of a targetpolynucleotide or polypeptide are known and available in the arts andinclude, e.g., methods employing reverse transcription-polymerase chainreaction (RT-PCR) and immunohistochemical techniques. In particularembodiments, the level of expression of a target polynucleotide orpolypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%,or greater than 50% as compared to an appropriate control value.

For example, if increased expression of a polypeptide desired, thenucleic acid may be an expression vector that includes a polynucleotidethat encodes the desired polypeptide. On the other hand, if reducedexpression of a polynucleotide or polypeptide is desired, then thenucleic acid may be, e.g., an antisense oligonucleotide, siRNA, ormicroRNA that comprises a polynucleotide sequence that specificallyhybridizes to a polynucleotide that encodes the target polypeptide,thereby disrupting expression of the target polynucleotide orpolypeptide. Alternatively, the nucleic acid may be a plasmid thatexpresses such an antisense oligonucleotide, siRNA, or microRNA.

In one particular embodiment, the present invention provides a method ofmodulating the expression of a polypeptide by a cell, comprisingproviding to a cell a lipid particle that consists of or consistsessentially of a lipid chosen from Table 1, DSPC, Chol and PEG-DMG orPEG-DMA, e.g., in a molar ratio of about 20-60% cationic lipid:5-25%DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA, wherein the lipid particleis associated with a nucleic acid capable of modulating the expressionof the polypeptide. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA). In another group of embodiments, the neutral lipid in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

In particular embodiments, the therapeutic agent is selected from ansiRNA, a microRNA, an antisense oligonucleotide, and a plasmid capableof expressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In related embodiments, the present invention provides a method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is selected from an siRNA, a microRNA, an antisenseoligonucleotide, and a plasmid capable of expressing an siRNA, amicroRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molar ratio ofabout 20-60% cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG orPEG-DMA, wherein the lipid particle is associated with the therapeuticnucleic acid. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA). In another group of embodiments, the neutral lipid in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molar ratio ofabout 20-60% cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG orPEG-DMA, wherein the lipid particle is associated with the therapeuticnucleic acid. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA). In another group of embodiments, the neutral lipid in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

The present invention further provides a method of inducing an immuneresponse in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is an immunostimulatory oligonucleotide. In certainembodiments, the immune response is a humoral or mucosal immuneresponse. In one embodiment, the pharmaceutical composition comprises alipid particle that consists of or consists essentially of a lipidchosen from Table 1, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molarratio of about 20-60% cationic lipid:5-25% DSPC:25-55% Chol:0.5-15%PEG-DMG or PEG-DMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In further embodiments, the pharmaceutical composition is provided tothe subject in combination with a vaccine or antigen. Thus, the presentinvention itself provides vaccines comprising a lipid particle of thepresent invention, which comprises an immunostimulatory oligonucleotide,and is also associated with an antigen to which an immune response isdesired. In particular embodiments, the antigen is a tumor antigen or isassociated with an infective agent, such as, e.g., a virus, bacteria, orparasite.

A variety of tumor antigens, infections agent antigens, and antigensassociated with other disease are well known in the art and examples ofthese are described in references cited herein. Examples of antigenssuitable for use in the present invention include, but are not limitedto, polypeptide antigens and DNA antigens. Specific examples of antigensare Hepatitis A, Hepatitis B, small pox, polio, anthrax, influenza,typhus, tetanus, measles, rotavirus, diphtheria, pertussis,tuberculosis, and rubella antigens. In a preferred embodiment, theantigen is a Hepatitis B recombinant antigen. In other aspects, theantigen is a Hepatitis A recombinant antigen. In another aspect, theantigen is a tumor antigen. Examples of such tumor-associated antigensare MUC-1, EBV antigen and antigens associated with Burkitt's lymphoma.In a further aspect, the antigen is a tyrosinase-related protein tumorantigen recombinant antigen. Those of skill in the art will know ofother antigens suitable for use in the present invention.

Tumor-associated antigens suitable for use in the subject inventioninclude both mutated and non-mutated molecules that may be indicative ofsingle tumor type, shared among several types of tumors, and/orexclusively expressed or overexpressed in tumor cells in comparison withnormal cells. In addition to proteins and glycoproteins, tumor-specificpatterns of expression of carbohydrates, gangliosides, glycolipids andmucins have also been documented. Exemplary tumor-associated antigensfor use in the subject cancer vaccines include protein products ofoncogenes, tumor suppressor genes and other genes with mutations orrearrangements unique to tumor cells, reactivated embryonic geneproducts, oncofetal antigens, tissue-specific (but not tumor-specific)differentiation antigens, growth factor receptors, cell surfacecarbohydrate residues, foreign viral proteins and a number of other selfproteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor p53 and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 andKSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionicgonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA)and melanocyte differentiation antigens such as Mart 1/Melan A, gp100,gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such asPSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene productssuch as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and othercancer testis antigens such as NY-ESO1, SSX2 and SCP1; mucins such asMuc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutralglycolipids and glycoproteins such as Lewis (y) and globo-H; andglycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.Also included as tumor-associated antigens herein are whole cell andtumor cell lysates as well as immunogenic portions thereof, as well asimmunoglobulin idiotypes expressed on monoclonal proliferations of Blymphocytes for use against B cell lymphomas.

Pathogens include, but are not limited to, infectious agents, e.g.,viruses, that infect mammals, and more particularly humans. Examples ofinfectious virus include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicularstomatitis viruses, rabies viruses); Coronaviridae (e.g.,coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae(e.g., parainfluenza viruses, mumps virus, measles virus, respiratorysyncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae(e.g., African swine fever virus); and unclassified viruses (e.g., theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcusfaecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfuenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Additional examples of pathogens include, but are not limited to,infectious fungi that infect mammals, and more particularly humans.Examples of infectious fingi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites include Plasmodium such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.Other infectious organisms (i.e., protists) include Toxoplasma gondii.

In one embodiment, the formulations of the invention can be used tosilence or modulate a target gene such as but not limited to FVII, Eg5,PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Dgene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, SORT1 gene, XBP1 gene, topoisomerase I gene,topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1)gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68gene, tumor suppressor genes, p53 tumor suppressor gene, p53 familymember DN-p63, pRb tumor suppressor gene, APC1 tumor suppressor gene,BRCA1 tumor suppressor gene, PTEN tumor suppressor gene, mLL fusiongene, BCR/ABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion gene,TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene, alphav-integrin gene, Flt-1 receptor gene, tubulin gene, Human PapillomaVirus gene, a gene required for Human Papilloma Virus replication, HumanImmunodeficiency Virus gene, a gene required for Human ImmunodeficiencyVirus replication, Hepatitis A Virus gene, a gene required for HepatitisA Virus replication, Hepatitis B Virus gene, a gene required forHepatitis B Virus replication, Hepatitis C Virus gene, a gene requiredfor Hepatitis C Virus replication, Hepatitis D Virus gene, a generequired for Hepatitis D Virus replication, Hepatitis E Virus gene, agene required for Hepatitis E Virus replication, Hepatitis F Virus gene,a gene required for Hepatitis F Virus replication, Hepatitis G Virusgene, a gene required for Hepatitis G Virus replication, Hepatitis HVirus gene, a gene required for Hepatitis H Virus replication,Respiratory Syncytial Virus gene, a gene that is required forRespiratory Syncytial Virus replication, Herpes Simplex Virus gene, agene that is required for Herpes Simplex Virus replication, herpesCytomegalovirus gene, a gene that is required for herpes Cytomegalovirusreplication, herpes Epstein Barr Virus gene, a gene that is required forherpes Epstein Barr Virus replication, Kaposi's Sarcoma-associatedHerpes Virus gene, a gene that is required for Kaposi'sSarcoma-associated Herpes Virus replication, JC Virus gene, human genethat is required for JC Virus replication, myxovirus gene, a gene thatis required for myxovirus gene replication, rhinovirus gene, a gene thatis required for rhinovirus replication, coronavirus gene, a gene that isrequired for coronavirus replication, West Nile Virus gene, a gene thatis required for West Nile Virus replication, St. Louis Encephalitisgene, a gene that is required for St. Louis Encephalitis replication,Tick-borne encephalitis virus gene, a gene that is required forTick-borne encephalitis virus replication, Murray Valley encephalitisvirus gene, a gene that is required for Murray Valley encephalitis virusreplication, dengue virus gene, a gene that is required for dengue virusgene replication, Simian Virus 40 gene, a gene that is required forSimian Virus 40 replication, Human T Cell Lymphotropic Virus gene, agene that is required for Human T Cell Lymphotropic Virus replication,Moloney-Murine Leukemia Virus gene, a gene that is required forMoloney-Murine Leukemia Virus replication, encephalomyocarditis virusgene, a gene that is required for encephalomyocarditis virusreplication, measles virus gene, a gene that is required for measlesvirus replication, Vericella zoster virus gene, a gene that is requiredfor Vericella zoster virus replication, adenovirus gene, a gene that isrequired for adenovirus replication, yellow fever virus gene, a genethat is required for yellow fever virus replication, poliovirus gene, agene that is required for poliovirus replication, poxvirus gene, a genethat is required for poxvirus replication, plasmodium gene, a gene thatis required for plasmodium gene replication, Mycobacterium ulceransgene, a gene that is required for Mycobacterium ulcerans replication,Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, agene that is required for Mycobacterium leprae replication,Staphylococcus aureus gene, a gene that is required for Staphylococcusaureus replication, Streptococcus pneumoniae gene, a gene that isrequired for Streptococcus pneumoniae replication, Streptococcuspyogenes gene, a gene that is required for Streptococcus pyogenesreplication, Chlamydia pneumoniae gene, a gene that is required forChlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Grog gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCAT gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.

DEFINITIONS

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

The term “acyl” refers to hydrogen, alkyl, partially saturated or fullysaturated cycloalkyl, partially saturated or fully saturatedheterocycle, aryl, and heteroaryl substituted carbonyl groups. Forexample, acyl includes groups such as (C1-C20)alkanoyl (e.g., formyl,acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),(C3-C20)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,benzo[b]thiophenyl-2-carbonyl, etc.).

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond. The term“alkylheterocyle” refers to a heteroaryl wherein at least one of thering atoms is substituted with alkyl, alkenyl or alkynyl

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio,alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl,arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl,haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino,alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl,aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonicacid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understoodthat the substituent may be further substituted. Exemplary substituentsinclude amino, alkylamino, dialkylamino, and cyclic amino compounds.

“Halogen” means fluoro, chloro, bromo and iodo.

The terms “alkylamine” and “dialkylamine” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively.

The term “alkylphosphate” refers to —O—P(Q′)(Q″)-O—R, wherein Q′ and Q″are each independently O, S, N(R)₂, optionally substituted alkyl oralkoxy; and R is optionally substituted alkyl, ω-aminoalkyl orω-(substituted)aminoalkyl.

The term “alkylphosphorothioate” refers to an alkylphosphate wherein atleast one of Q′ or Q″ is S.

The term “alkylphosphonate” refers to an alkylphosphate wherein at leastone of Q′ or Q″ is alkyl.

The term “hydroxyalkyl” means —O-alkyl radical.

The term “alkylheterocycle” refers to an alkyl where at least onemethylene has been replaced by a heterocycle.

The term “ω-aminoalkyl” refers to -alkyl-NH₂ radical. And the term“ω-(substituted)aminoalkyl refers to an ω-aminoalkyl wherein at leastone of the H on N has been replaced with alkyl.

The term “ω-phosphoalkyl” refers to -alkyl-O—P(Q′)(Q″)-O—R, wherein Q′and Q″ are each independently O or S and R optionally substituted alkyl.

The term “ω-thiophosphoalkyl refers to ω-phosphoalkyl wherein at leastone of Q′ or Q” is S.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et. al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples.

EXAMPLES Example 1 Synthesis of methanesulfonic acid octadeca-9,12-dienyl ester 2

To a solution of the alcohol 1 (26.6 g, 100 mmol) in dichloromethane(100 mL), triethylamine (13.13 g, 130 mmol) was added and this solutionwas cooled in an ice-bath. To this cold solution, a solution of mesylchloride (12.6 g, 110 mmol) in dichloromethane (60 mL) was addeddropwise and after the completion of the addition, the reaction mixturewas allowed to warm to ambient temperature and stirred overnight. TheTLC of the reaction mixture showed the completion of the reaction. Thereaction mixture was diluted with dichloromethane (200 mL), washed withwater (200 mL), satd. NaHCO₃ (200 mL), brine (100 mL) and dried (NaSO₄).The organic layer was concentrated to get the crude product which waspurified by column chromatography (silica gel) using 0-10% Et₂O inhexanes. The pure product fractions were combined and concentrated toobtain the pure product 2 as colorless oil (30.6 g, 89%). ¹H NMR (CDCl₃,400 MHz) δ=5.42-5.21 (m, 4H), 4.20 (t, 2H), 3.06 (s, 3H), 2.79 (t, 2H),2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H), 1.06-1.18 (m, 18H), 0.88 (t, 3H).¹³C NMR (CDCl₃) δ=130.76, 130.54, 128.6, 128.4, 70.67, 37.9, 32.05,30.12, 29.87, 29.85, 29.68, 29.65, 29.53, 27.72, 27.71, 26.15, 25.94,23.09, 14.60. MS. Molecular weight calculated for C₁₉H₃₆O₃S, Cal.344.53. Found 343.52 (M−H⁻).

Synthesis of 18-Bromo-octadeca-6, 9-diene 3

The mesylate 2 (13.44 g, 39 mmol) was dissolved in anhydrous ether (500mL) and to it the MgBr.Et₂O complex (30.7 g, 118 mmol) was added underargon and the mixture was refluxed under argon for 26 h after which theTLC showed the completion of the reaction. The reaction mixture wasdiluted with ether (200 mL) and ice-cold water (200 mL) was added tothis mixture and the layers were separated. The organic layer was washedwith 1% aqueous K₂CO₃ (100 mL), brine (100 mL) and dried (Anhyd.Na₂SO₄). Concentration of the organic layer provided the crude productwhich was further purified by column chromatography (silica gel) using0-1% Et₂O in hexanes to isolate the bromide 3 (12.6 g, 94%) as acolorless oil. ¹H NMR (CDCl₃, 400 MHz) δ=5.41-5.29 (m, 4H), 4.20 (d,2H), 3.40 (t, J=7 Hz, 2H), 2.77 (t, J=6.6 Hz, 2H), 2.09-2.02 (m, 4H),1.88-1.00 (m, 2H), 1.46-1.27 (m, 18H), 0.88 (t, J=3.9 Hz, 3H). ¹³C NMR(CDCl₃) δ=130.41, 130.25, 128.26, 128.12, 34.17, 33.05, 31.75, 29.82,29.57, 29.54, 29.39, 28.95, 28.38, 27.42, 27.40, 25.84, 22.79, 14.28.

Synthesis of 18-Cyano-octadeca-6, 9-diene 4

To a solution of the mesylate (3.44 g, 10 mmol) in ethanol (90 mL), asolution of KCN (1.32 g, 20 mmol) in water (10 mL) was added and themixture was refluxed for 30 min. after which, the TLC of the reactionmixture showed the completion of the reaction after which, ether (200mL) was added to the reaction mixture followed by the addition of water.The reaction mixture was extracted with ether and the combined organiclayers was washed with water (100 mL), brine (200 mL) and dried.Concentration of the organic layer provided the crude product which waspurified by column chromatography (0-10% Et₂O in hexanes). The pureproduct 4 was isolated as colorless oil (2 g, 74%). ¹H NMR (CDCl₃, 400MHz) δ=5.33-5.22 (m, 4H), 2.70 (t, 2H), 2.27-2.23 (m, 2H), 2.00-1.95 (m,4H), 1.61-1.54 (m, 2H), 1.39-1.20 (m, 18H), 0.82 (t, 3H). ¹³C NMR(CDCl₃) δ=130.20, 129.96, 128.08, 127.87, 119.78, 70.76, 66.02, 32.52,29.82, 29.57, 29.33, 29.24, 29.19, 29.12, 28.73, 28.65, 27.20, 27.16,25.62, 25.37, 22.56, 17.10, 14.06. MS. Molecular weight calculated forC₁₉H₃₃N, Cal. 275.47. Found 276.6 (M−H⁻).

Synthesis of Heptatriaconta-6,9,28,31-tetraen-19-one 7

To a flame dried 500 mL 2NRB flask, freshly activated Mg turnings (0.144g, 6 mmol) were added and the flask was equipped with a magnetic stirbar and a reflux condenser. This set-up was degassed, flushed with argonand 10 mL of anhydrous ether was added to the flask via syringe. Thebromide 3 (1.65 g, 5 mmol) was dissolved in anhydrous ether (10 mL) andadded dropwise via syringe to the flask. An exothermic reaction wasnoticed (to confirm/accelerate the Grignard reagent formation, 2 mg ofiodine was added and immediate decolorization was observed confirmingthe formation of the Grignard reagent) and the ether started refluxing.After the completion of the addition the reaction mixture was kept at35° C. for 1 h and then cooled in ice bath. The cyanide 4 (1.38 g, 5mmol) was dissolved in anhydrous ether (20 mL) and added dropwise to thereaction mixture with stirring. An exothermic reaction was observed andthe reaction mixture was stirred overnight at ambient temperature. Thereaction was quenched by adding 10 mL of acetone dropwise followed byice cold water (60 mL). The reaction mixture was treated with aq. H₂SO₄(10% by volume, 200 mL) until the solution became homogeneous and thelayers were separated. The aq. phase was extracted with ether (2×100mL). The combined ether layers were dried (Na₂SO₄) and concentrated toget the crude product which was purified by column (silica gel, 0-10%ether in hexanes) chromatography. The pure product fractions wereevaporated to provide the pure ketone 7 as a colorless oil (2 g, 74%).¹H NMR (CDCl₃, 400 MHz) δ=5.33-5.21 (m, 8H), 2.69 (t, 4H), 2.30 (t, 4H),2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m, 18H), 0.82 (t, 3H).¹³C NMR (CDCl₃) δ=211.90, 130.63, 130.54, 128.47, 128.41, 43.27, 33.04,32.01, 30.93, 29.89, 29.86, 29.75, 29.74, 27.69, 26.11, 24.35, 23.06,14.05. MS. Molecular weight calculated for C₃₇H₆₆O, Cal. 526.92. Found528.02 (M+H⁺).

Example 2. Alternative Synthesis of the Ketone 7

Synthesis of Compound 6b

To a flame dried 500 mL RB flask, freshly activated Mg turnings (2.4 g,100 mmol) were added and the flask was equipped with a magnetic stirbar, an addition funnel and a reflux condenser. This set-up was degassedand flushed with argon and 10 mL of anhydrous ether was added to theflask via syringe. The bromide 3 (26.5 g, 80.47 mmol) was dissolved inanhydrous ether (50 mL) and added to the addition funnel. About 5 mL ofthis ether solution was added to the Mg turnings while stirringvigorously. An exothermic reaction was noticed (to confirm/acceleratethe Grignard reagent formation, 5 mg of iodine was added and immediatedecolorization was observed confirming the formation of the Grignardreagent) and the ether started refluxing. The rest of the solution ofthe bromide was added dropwise while keeping the reaction under gentlereflux by cooling the flask in water. After the completion of theaddition the reaction mixture was kept at 35° C. for 1 h and then cooledin ice bath. Ethyl formate (2.68 g, 36.2 mmol) was dissolved inanhydrous ether (40 mL) and transferred to the addition funnel and addeddropwise to the reaction mixture with stirring. An exothermic reactionwas observed and the reaction mixture started refluxing. After theinitiation of the reaction the rest of the ethereal solution of formatewas quickly added as a stream and the reaction mixture was stirred for afurther period of 1 h at ambient temperature. The reaction was quenchedby adding 10 mL of acetone dropwise followed by ice cold water (60 mL).The reaction mixture was treated with aq. H₂SO₄ (10% by volume, 300 mL)until the solution became homogeneous and the layers were separated. Theaq. phase was extracted with ether (2×100 mL). The combined ether layerswere dried (Na₂SO₄) and concentrated to get the crude product which waspurified by column (silica gel, 0-10% ether in hexanes) chromatography.The slightly less polar fractions were concentrated to get the formate6a (1.9 g) and the pure product fractions were evaporated to provide thepure product 6b as a colorless oil (14.6 g, 78%).

Synthesis of Compound 7

To a solution of the alcohol 6b (3 g, 5.68 mmol) in CH₂Cl₂ (60 mL),freshly activated 4 A molecular sieves (50 g) were added and to thissolution powdered PCC (4.9 g, 22.7 mmol) was added portion wise over aperiod of 20 minutes and the mixture was further stirred for 1 hour(Note: careful monitoring of the reaction is necessary in order to getgood yields since prolonged reaction times leads to lower yields) andthe TLC of the reaction mixture was followed every 10 minutes (5% etherin hexanes) After completion of the reaction, the reaction mixture wasfiltered through a pad of silica gel and the residue was washed withCH₂Cl₂ (400 mL). The filtrate was concentrated and the thus obtainedcrude product was further purified by column chromatography (silica gel,1% Et₂O in hexanes) to isolate the pure product 7 (2.9 g, 97%) as acolorless oil. ¹H NMR (CDCl₃, 400 MHz) δ=5.33-5.21 (m, 8H), 2.69 (t,4H), 2.30 (t, 4H), 2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m,18H), 0.82 (t, 3H). ¹³C NMR (CDCl₃) δ=211.90, 130.63, 130.54, 128.47,128.41, 43.27, 33.04, 32.01, 30.93, 29.89, 29.86, 29.75, 29.74, 27.69,26.11, 24.35, 23.06, 14.05. MS. Molecular weight calculated for C₃₇H₆₆O,Cal. 526.92. Found 528.02 (M+H⁺).

Example 3. Synthesis of Unsymmetric Ketones 25 and 27

Synthesis of heptatriaconta-6,9,28-trien-19-one 25

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (132 mg,0.0054 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide 24 (1.8 g, 0.0054 mol) was dissolved in anhydrousether (10 mL) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide 4 (0.5 g, 0.0018 mol) was dissolved in dry THF (5 mL) andadded dropwise to the reaction with stirring. An exothermic reaction wasobserved and the reaction mixture was refluxed (at 70° C.) for 12 h andquenched with ammonium chloride solution. It was then treated with 25%HCl solution until the solution became homogenous and the layers wereseparated. The aqueous phase was extracted with ether. The combinedether layers were dried and concentrated to get the crude product whichwas purified by column chromatography. The pure product fractions wereevaporated to provide the pure ketone 25 as colorless oil.

Yield: 0.230 g (24%). ¹H-NMR (CDCl3, 400 MHz): δ=5.37-5.30 (m, 6H),2.77-2.74 (t, 2H), 2.38-2.34 (t, 4H), 2.05-1.95 (m, 8H), 1.56-1.52 (m,4H), 1.35-1.25 (m, aliphatic protons), 0.89-0.85 (t, 6H). IR(cm-1):2924, 2854, 1717, 1465, 1049, 721.

Synthesis of heptatriaconta-6,9-dien-19-one 27

To a flame dried 500 mL 2NRB flask, a freshly activated Mg turnings(0.144 g, 6 mmol) is added and the flask is equipped with a magneticstir bar and a reflux condenser. This set-up is degassed and flushedwith argon and 10 mL of anhydrous ether is added to the flask viasyringe. The commercially available bromide 26 (2.65 g, 5 mmol) isdissolved in anhydrous ether (10 mL) and added dropwise via syringe tothe flask. After the completion of the addition the reaction mixture iskept at 35° C. for 1 h and then cooled in ice bath. The cyanide 4 (1.38g, 5 mmol) is dissolved in anhydrous ether (20 mL) and added dropwise tothe reaction mixture with stirring. An exothermic reaction is observedand the reaction mixture is stirred overnight at ambient temperature.The reaction is quenched by adding 10 mL of acetone dropwise followed byice cold water (60 mL) The reaction mixture is treated with aq. H₂SO₄(10% by volume, 200 mL) until the solution becomes homogeneous and thelayers are separated. The aq. phase is extracted with ether (2×100 mL).The combined ether layers are dried (Na₂SO₄) and concentrated to get thecrude product which is purified by column chromatography to provide thepure ketone 27 as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=5.42-5.30(m, 4H), 2.79-2.78 (t, 2H), 2.40-2.37 (t, 4H), 2.08-2.03 (m, 4H),1.58-1.54 (m, 4H), 1.36-1.26 (br m, aliphatic protons), 0.91-0.87 (t,6H). IR (cm-1):2924, 2854, 1716, 1465, 1375, 721.

Example 4. Synthesis of Unsymmetrical Ketones with C₁₂ Chain

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (175 mg,0.0072 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide 28 (1.5 g, 0.006 mol) was dissolved in anhydrousether (7 ml) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide 4 (1 g, 0.0036 mol) was dissolved in anhydrous ether (7mL) and added dropwise to the reaction with stirring. An exothermicreaction was observed and the reaction mixture was refluxed for 12 h andquenched with ammonium chloride solution. It was then treated with 25%HCl solution until the solution becomes homogenous and the layers wereseparated. The aq phase was extracted with ether. The combined etherlayers were dried and concentrated to get the crude product which waspurified by column chromatography. The pure product fractions wereevaporated to provide the pure ketone 29 as colorless oil. Yield: 0.65 g(26%). ¹H-NMR (δ ppm): 5.388-5.302 (m, 4H), 2.77-2.74 (t, 2H), 2.38-2.34(t, 4H), 2.04-2.01 (m, 4H), 1.34-1.18 (m, 36H), 0.89-0.85 (m 6H). IR(cm⁻¹): 3009, 2920, 2851, 1711 (C═O), 1466, 1376, 1261.

Example 5. Synthesis of Unsymmetrical Ketones with C₁₀ Chain 31

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (266 mg,0.0109 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide (2.43 g, 0.0109 mol) was dissolved in anhydrousether (7 ml) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide (1 g, 0.0036 mol) was dissolved in anhydrous ether (7 mL)and added dropwise to the reaction with stirring. An exothermic reactionwas observed and the reaction mixture was stirred at ambient temperaturefor 2 hr. THF (4 ml) was added to the reaction mixture and it was warmedto 45-50° C. for 4 hr till the cyano derivative was complete consumed.The reaction was quenched by adding 3 mL of acetone dropwise followed byice cold water. The reaction mixture was treated with 25% HCl solutionuntil the solution becomes homogenous and the layers were separated. Theaq. phase was extracted with ether. The combined ether layers were driedand concentrated to get the crude product which was purified by columnchromatography. The pure product fractions were evaporated to providethe pure ketone as colorless oil. Yield: 0.93 gms (61%). ¹H-NMR (δ ppm):5.37-5.302 (m, 4H), 2.77-2.74 (t, 2H), 2.38-2.34 (t, 4H), 2.05-2.00 (m,4H), 1.55-1.52 (m, 2H), 1.35-1.24 (m, 34H), 0.89-0.84 (m 6H). IR (cm⁻¹):3009, 2925, 2854, 1717 (C═O), 1465, 1376.

Example 6. Synthesis of Unsymmetrical Ketones with Cholesterol 33

Using a similar procedure to that used for the synthesis of ketone 31,the cholesteryl chloride on conversion to the corresponding magnesiumchloride followed by addition to the linoleyl cyanide provided theketone 33.

Example 7. Synthesis of Unsymmetrical Ketones with Cholesterol 35

The treatment of the cholesterolchloroformate with 3-bromopropylamineprovided the bromide 34 which is converted to the corresponding Grignardreagent 34a which on treatment with the linoleyl cyanide provided thecorresponding unsymmetrical ketone 35 in good yield.

Example 8. Synthesis of Unsymmetric Ketone 40

Synthesis of Compound 37

To a 500 ml two neck RBF containing LiAlH₄ (1.02 g, 0.0269 mol) wasadded anhydrous THF (20 mL) at room temperature under nitrogenatmosphere. The suspension was stirred for 1 h at room temperature andthen cooled to 0° C. To this mixture was added a solution of compound 1(5 g, 0.01798 mol) in anhydrous THF (50 mL) slowly maintaining theinside temperature 0° C. After completion of the addition, reactionmixture was warmed to ambient temperature and stirred for 1 h. Progressof the reaction was monitored by TLC. Upon completion of the reaction,mixture was cooled to 0° C. and quenched with sat. solution of aq.Na₂SO₄. Reaction mixture was stirred for 30 minutes and solid formed wasfiltered through celite bed and washed with ethyl acetate (100 mL)Filtrate and washings were combined and evaporated on rotary evaporatorto afford the compound 37 as colorless liquid, which was taken as suchfor the next stage without any purification. Yield: (4.5 g, 95%); ¹H NMR(400 MHz, CDCl₃) δ=5.39-5.28 (m, 6H), 3.64-3.61 (t, 2H), 2.81-2.78 (t,4H), 2.10-2.01 (m, 4H), 1.59-1.51 (m, 2H), 1.29-1.22 (m, aliphaticprotons), 0.98-0.94 (t, 3H).

Synthesis of Compound 38

Compound 37 (14 g, 0.0530 mol) was dissolved in DCM (300 ml) in a 500 mltwo neck RBF and cooled to 0° C. To this solution was addedtriethylamine (29.5 ml, 0.2121 mol) slowly under inert atmosphere.Reaction mixture was then stirred for 10-15 minutes and to it mesylchloride (6.17 mL, 0.0795 mol)) was added slowly. After completeaddition, the reaction mixture was allowed to warm to ambienttemperature and stirred for 20 h. Reaction was monitored by TLC. Uponcompletion, the reaction mixture was diluted with water (200 mL) stirredfor few minutes and organic layer was separated. Organic phase wasfurther washed with brine (1×70 mL), dried over Na₂SO₄. and solvent wasremoved on rotary evaporator to get the crude compound 38 as brown oilwhich was used as such for next reaction. Yield: (17 g, 93%) ¹H NMR (400MHz, CDCl₃) δ=5.39-5.31 (m, 6H), 4.22-4.19 (t, 2H), 2.99 (s, 3H),2.81-2.78 (m, 4H), 2.08-2.01 (m, 4H), 1.75.1.69 (m, 2H), 1.39-1.29 (m,aliphatic protons), 0.98-0.94 (t, 3H).

Synthesis of Compound 39

The mesylate 38 (10 g, 0.2923 mol) was dissolved in (300 mL) anhydrousether in a 1000 mL two neck RBF and MgBr₂.Et₂O complex (22.63 g, 0.0877mol) was added into it under nitrogen atmosphere. Resulting mixture wasthen heated to reflux for 26 h. After completion of the reaction (byTLC), reaction mixture was diluted with ether (300 mL) and ice coldwater (200 mL) and ether layer was separated out. Organic layer was thenwashed with 1% aq. K₂CO₃ (100 mL) followed by brine (80 mL). Organicphase was then dried over anhydrous Na₂SO₄ and solvent was evaporatedoff under vacuum to give the crude material which was chromatographed onsilca gel (60-120 mesh) using 0-1% ethyl acetate in hexane as elutingsystem to yield the desired compound 39 as oil. Yield: (7 g, 73%) ¹H NMR(400 MHz, CDCl₃) δ=5.39-5.31 (m, 6H), 3.41-3.37 (t, 2H), 2.81-2.78 (m,4H), 2.08-2.02 (m, 4H), 1.86-1.80 (m, 2H), 1.42-1.29 (m, aliphaticprotons), 0.98-0.94 (t, 3H).

Synthesis of Unsymetric Ketone 40

To a flame dried 500 mL two neck RBF, equipped with magnetic stir barand a reflux condenser, freshly activated Mg turnings (0.88 g, 0.03636mol) were added. This set up was degassed, flushed with argon and ether(150 mL) was added into it. Few drops of bromo compound 4 (11.89 g,0.03636 mol) in 50 mL ether was added at the beginning to initiate thereaction (note: catalytic amount of 1, 2-dibromo ethane was also addedto accelerate formation of grignard reagent). Upon initiation, theremaining solution of bromo compound was added slowly to the refluxingethereal solution. After complete addition, the reaction mixture wasrefluxed at 40° C. for 1.5 h. It was then cooled to 10° C. and thelinoleyl cyanide 4 (5 g, 0.01818 mol) in 30 mL of dry ether was addeddrop wise and the resulting mixture was then heated to reflux for 20 hat 40° C. Progress of the reaction was monitored by TLC. After completeconsumption of the cyano derivative 40 (by TLC), mixture was cooled toroom temperature and quenched with 30 mL of acetone followed by (50 mL)ice water. This solution was further acidified with 10% HCl solution andether layer was separated out. Aqueous phase was further extracted withdiethyl ether (2×100 mL). Removal of the solvent after drying overanhydrous Na₂SO₄ afforded the crude ketone which was purified by silicagel column chromatography (100-200 mesh) using 0-5% ether in hexane aseluting system to give the title compound 40 as pale yellow oil. Yield:(4.8 g, 50.5%) ¹H NMR (400 MHz, CDCl₃) δ=5.38-5.28 (m, 10H), 2.80-2.74(m, 6H), 2.38-2.34 (t, 4H), 2.08-2.00 (m, 8H), 1.55-1.52 (m, 4H),1.35-1.26 (m, aliphatic protons), 0.98-0.94 (t, 3H), 0.89-0.85 (t, 3H).HPLC—98.04%.

Example 9. Oligonucleotide Synthesis

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG,500°A, Prime Synthesis) and RNA phosphoramidites with standardprotecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

3′-ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence was performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol was tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled siRNAs were synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite were purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block Anextended 15 min coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate was introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent The cholesterol phosphoramidite was synthesized in house, andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite was 16 minutes.

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dyince and dried under vacuum on a speed vac.

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 min and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLC analysis

TABLE 7 siRNA duplexes for Luc and FVII targeting. SEQ DuplexSense/Antisense Sequence 5′-3′ ID Target 1000/2434 CUU ACG CUG AGU ACUUCG AdTdT NO: 61 Luc U*CG AAG fUAC UCA GCG fUAA GdT*dT NO: 62 2433/1001C*UfU ACG CUG AGfU ACU UCG AdT*dT NO: 63 Luc UCG AAG UAC UCA GCG UAAGdTdT NO: 64 2433/2434 C*UfU ACG CUG AGfU ACU UCG AdT*dT NO: 63 Luc U*CGAAG fUAC UCA GCG fUAA GdT*dT NO: 62 1000/1001 CUU ACG CUG AGU ACU UCGAdTdT NO: 61 Luc UCG AAG UAC UCA GCG UAA GdTdT NO: 64 AD-GGAUCAUCUCAAGUCUUACdTdT NO: 65 FVII 1596 GUAAGACUUGAGAUGAUCCdTdT NO: 66AD- GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdTsdT NO: 67 FVII 1661GfUAAGAfCfUfUGAGAfUGAfUfCfCdT*dT NO: 68 Note:

lowercase is 2′-O-methyl modified nucleotide, *is phosphorothioatebackbone linkages, fN is a 2′-fluoro nucleotide, dN is 2′-deoxynucleotide.

Example 10. Serum Stability Assay for siRNA

A medium throughput assay for initial sequence-based stability selectionwas performed by the “stains all” approach. To perform the assay, ansiRNA duplex was incubated in 90% human serum at 37° C. Samples of thereaction mix were quenched at various time points (at 0 min., 15, 30,60, 120, and 240 min.) and subjected to electrophoretic analysis (FIG.1). Cleavage of the RNA over the time course provided informationregarding the susceptibility of the siRNA duplex to serum nucleasedegradation.

A radiolabeled dsRNA and serum stability assay was used to furthercharacterize siRNA cleavage events. First, a siRNA duplex was5′end-labeled with ³²P on either the sense or antisense strand. Thelabeled siRNA duplex was incubated with 90% human serum at 37° C., and asample of the solution was removed and quenched at increasing timepoints. The samples were analyzed by electrophoresis.

Example 11: FVII In Vivo Evaluation Using the Cationic Lipid DerivedLiposomes

In Vivo Rodent Factor VII and ApoB Silencing Experiments.

C57BL/6 mice (Charles River Labs, MA) and Sprague-Dawley rats (CharlesRiver Labs, MA) received either saline or siRNA in desired formulationsvia tail vein injection at a volume of 0.01 mL/g. At various time pointspost-administration, animals were anesthesized by isofluorane inhalationand blood was collected into serum separator tubes by retro orbitalbleed. Serum levels of Factor VII protein were determined in samplesusing a chromogenic assay (Coaset Factor VII, DiaPharma Group, OH orBiophen FVII, Aniara Corporation, OH) according to manufacturerprotocols. A standard curve was generated using serum collected fromsaline treated animals. In experiments where liver mRNA levels wereassessed, at various time points post-administration, animals weresacrificed and livers were harvested and snap frozen in liquid nitrogen.Frozen liver tissue was ground into powder. Tissue lysates were preparedand liver mRNA levels of Factor VII and apoB were determined using abranched DNA assay (QuantiGene Assay, Panomics, Calif.).

Example 12. Preparation of 1,2-Di-O-alkyl-sn3-Carbomoylglyceride(PEG-DMG)

Preparation of IVa

1,2-Di-O-tetradecyl-sn-glyceride Ia (30 g, 61.80 mmol) andN,N′-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) were taken indichloromethane (DCM, 500 mL) and stirred over an ice water mixture.Triethylamine (TEA, 25.30 mL, 3 eq) was added to the stirring solutionand subsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard work-up. The residue obtained was dried at ambienttemperature under high vacuum overnight. After drying the crudecarbonate IIa thus obtained was dissolved in dichloromethane (500 mL)and stirred over an ice bath. To the stirring solution mPEG₂₀₀₀-NH₂(III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) andanhydrous pyridine (Py, 80 mL, excess) were added under argon. Thereaction mixture was then allowed to stir at ambient temperatureovernight. Solvents and volatiles were removed under vacuum and theresidue was dissolved in DCM (200 mL) and charged on a column of silicagel packed in ethyl acetate. The column was initially eluted with ethylacetate and subsequently with gradient of 5-10% methanol indichloromethane to afford the desired PEG-Lipid IVa as a white solid(105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz) □=5.20-5.12 (m, 1H), 4.18-4.01(m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂),2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15 (m,48H), 0.84 (t, 6.5 Hz, 6H). MS range found: 2660-2836.

Preparation of IVb

1,2-Di-O-hexadecyl-sn-glyceride Ib (1.00 g, 1.848 mmol) and DSC (0.710g, 1.5 eq) were taken together in dichloromethane (20 mL) and cooleddown to 0° C. in an ice water mixture. Triethylamine (1.00 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solutionand dried over sodium sulfate. Solvents were removed under reducedpressure and the resulting residue of IIb was maintained under highvacuum overnight. This compound was directly used for the next reactionwithout further purification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol,purchased from NOF Corporation, Japan) and IIb (0.702 g, 1.5 eq) weredissolved in dichloromethane (20 mL) under argon. The reaction wascooled to 0° C. Pyridine (1 mL, excess) was added and the reactionstirred overnight. The reaction was monitored by TLC. Solvents andvolatiles were removed under vacuum and the residue was purified bychromatography (first ethyl acetate followed by 5-10% MeOH/DCM as agradient elution) to obtain the required compound IVb as a white solid(1.46 g, 76%). ¹H NMR (CDCl₃, 400 MHz) δ=5.17 (t, J=5.5 Hz, 1H), 4.13(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz, 1H),3.82-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.05-1.90 (m,2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m, 56H), 0.85 (t,J=6.5 Hz, 6H). MS range found: 2716-2892.

Preparation of IVc

1,2-Di-O-octadecyl-sn-glyceride Ic (4.00 g, 6.70 mmol) and DSC (2.58 g,1.5 eq) were taken together in dichloromethane (60 mL) and cooled downto 0° C. in an ice water mixture. Triethylamine (2.75 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solution,and dried over sodium sulfate. Solvents were removed under reducedpressure and the residue was maintained under high vacuum overnight.This compound was directly used for the next reaction without furtherpurification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and IIc (0.760 g, 1.5 eq) were dissolved indichloromethane (20 mL) under argon. The reaction was cooled to 0° C.Pyridine (1 mL, excess) was added and the reaction was stirredovernight. The reaction was monitored by TLC. Solvents and volatileswere removed under vacuum and the residue was purified by chromatography(ethyl acetate followed by 5-10% MeOH/DCM as a gradient elution) toobtain the desired compound IVc as a white solid (0.92 g, 48%). ¹H NMR(CDCl₃, 400 MHz) δ=5.22-5.15 (m, 1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz,1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75 (m, 2H), 3.70-3.20(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70 (m, 2H), 1.60-1.48 (m, 4H),1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2774-2948.

Example 13

Synthesis of 2005

To a solution of 2004 (50 g, 95 mmol) in DCM (400 ml) under Aratmosphere, was added TEA (53 mL, 378 mmol) and DMAP (1.2 g, 9.5 mmol)and stirred at room temperature under Ar atmosphere. Reaction mass wascooled to −5° C. and the solution of mesyl chloride (15 mL, 190 mmol) inDCM (100 ml) was added slowly at temperature below −5° C. and allowed towarm to RT after addition. After 30 minutes (TLC), reaction mass wasquenched with ice cold water (20 ml). Organic layer was separated,washed with 1N HCl (30 ml), water, brine, dried over sodium sulfate andevaporated at reduced pressure to obtain pure product (55 g, 95.5%) asyellow liquid. 1H NMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J=6.8), 1.2-1.5(m, 36H), 1.67 (m, 4H), 2.05 (q, 8H, J1=6.8, J2=6.8), 2.77 (t, 4H,J=6.4), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m, 8H).

Synthesis 2006

To a solution of 2005 (50 g, 82 mmol) in DMF (500 mL) under argonatmosphere, was added NaN₃ (27 g, 410 mmol) and heated to 70° C. andmaintained the temperature for four hours (TLC). The mixture was dilutedwith water and extracted with ethyl acetate (3×250 ml). The organiclayer was washed with water, brine, dried over Na₂SO₄ and evaporated atreduced pressure to give crude product, which was purified by silica gelchromatography using hexane/ether as eluent. The product was eluted at2% ether hexane to get 2006 (36 g, 86%) as pale yellow liquid. ¹H NMR(400 MHz, CDCl3): δ 0.90 (t, 8H), 1.30 (m, 36H), 1.49 (t, 4H, J=6.4 Hz)2.04 (q, 8H, J1=7.6, J2=14 Hz), 2.77 (t, 4H, J=6.4 Hz), 3.22 (m, 1H),5.34 (m, 8H). ¹³C NMR (400 MHz, CDCl₃): δ 14.1, 22.5, 25.6, 26.1, 27.2,29.2, 29.3, 29.45, 29.65, 31.5, 34.1, 63.1, 127.9, and 130.1. IR (KBr):2098.

Example 14: siRNA Formulation Using Preformed Vesicles

Cationic lipid containing particles were made using the preformedvesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid weresolubilised in ethanol at a molar ratio of 40/10/40/10, respectively.The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4)with mixing to a final ethanol and lipid concentration of 30% (vol/vol)and 6.1 mg/mL respectively and allowed to equilibrate at roomtemperature for 2 min before extrusion. The hydrated lipids wereextruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22°C. using a Lipex Extruder (Northern Lipids, Vancouver, BC) until avesicle diameter of 70-90 nm, as determined by Nicomp analysis, wasobtained. This generally required 1-3 passes. For some cationic lipidmixtures which did not form small vesicles hydrating the lipid mixturewith a lower pH buffer (50 mM citrate, pH 3) to protonate the phosphategroup on the DSPC headgroup helped form stable 70-90 nm vesicles.

The FVII siRNA (solubilised in a 50 mM citrate, pH 4 aqueous solutioncontaining 30% ethanol) was added to the vesicles, pre-equilibrated to35° C., at a rate of ˜5 mL/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was achieved, the mixture wasincubated for a further 30 min at 35° C. to allow vesiclere-organization and encapsulation of the FVII siRNA. The ethanol wasthen removed and the external buffer replaced with PBS (155 mM NaCl, 3mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by either dialysis or tangential flowdiafiltration. The final encapsulated siRNA-to-lipid ratio wasdetermined after removal of unencapsulated siRNA using size-exclusionspin columns or ion exchange spin columns.

Example 15: In Vivo Determination of Efficacy of Novel LipidFormulations

Test formulations were initially assessed for their FVII knockdown infemale 7-9 week old, 15-25 g, female C57Bl/6 mice at 0.1, 0.3, 1.0 and5.0 mg/kg with 3 mice per treatment group. All studies included animalsreceiving either phosphate-buffered saline (PBS, Control group) or abenchmark formulation. Formulations were diluted to the appropriateconcentration in PBS immediately prior to testing. Mice were weighed andthe appropriate dosing volumes calculated (10 μl/g body weight). Testand benchmark formulations as well as PBS (for Control animals) wereadministered intravenously via the lateral tail vein. Animals wereanesthetised 24 h later with an intraperitoneal injection ofKetamine/Xylazine and 500-700 μl of blood was collected by cardiacpuncture into serum separator tubes (BD Microtainer). Blood wascentrifuged at 2,000×g for 10 min at 15° C. and serum was collected andstored at −70° C. until analysis. Serum samples were thawed at 37° C.for 30 min, diluted in PBS and aliquoted into 96-well assay plates.Factor VII levels were assessed using a chromogenic assay (Biophen FVIIkit, Hyphen BioMed) according to manufacturer's instructions andabsorbance measured in microplate reader equipped with a 405 nmwavelength filter. Plasma FVII levels were quantified and ED50s (doseresulting in a 50% reduction in plasma FVII levels compared to controlanimals) calculated using a standard curve generated from a pooledsample of serum from Control animals. Those formulations of interestshowing high levels of FVII knockdown (ED50<<0.1 mg/kg) were re-testedin independent studies at a lower dose range to confirm potency andestablish ED50.

FIG. 3 provides a Table depicting the EC50 of exemplary compounds testedusing this method.

Example 15A: Determination of pKa of Formulated Lipids

The pKa's of the different ionisable cationic lipids were determinedessentially as described (Eastman et al 1992 Biochemistry 31:4262-4268)using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid(TNS), which is non-fluorescent in water but becomes appreciablyfluorescent when bound to membranes. Vesicles composed of cationiclipid/DSPC/CH/PEG-c-DOMG (40:10:40:10 mole ratio) were diluted to 0.1 mMin buffers (130 mM NaCl, 10 mM CH₃COONH₄, 10 mM MES, 10 mM HEPES) ofvarious pH's, ranging from 2 to 11. An aliquot of the TNS aqueoussolution (1 μM final) was added to the diluted vesicles and after a 30second equilibration period the fluorescent of the TNS-containingsolution was measured at excitation and emission wavelengths of 321 nmand 445 nm, respectively. The pKa of the cationic lipid-containingvesicles was determined by plotting the measured fluorescence againstthe pH of the solutions and fitting the data to a Sigmodial curve usingthe commercial graphing program IgorPro.

FIG. 3 provides a Table depicting the pKa of exemplary compounds testedusing this method.

Example 16: Synthesis of Guanidinium Linked Lipids Guanidinium AnalogsPreparation of Compound 7204

Preparation of Compound 7013

To a mixture of 1,2,4-butanetriol (7012, 21.2 g, 200 mmol, 5.0 eq),dilinoleyl ketone (21.0 g, 40.0 mmol, 1.0 eq) and p-toluenesulfonic acid(0.76 g, 4.0 mmol, 0.1 eq) in toluene was refluxed under Dean-stockconditions for overnight. After completion of the reaction, was cooled,evaporated the solvent and purified by column chromatography usinghexane and ethyl acetate (15%) as gradients gave desired ketal (7013) in47% yield as an oil. ¹H NMR (400 MHz, CDCl₃) δ 5.48-5.24 (m, 8H),4.32-4.17 (m, 1H), 4.08 (dd, J=7.8, 6.1, 1H), 3.86-3.74 (m, 2H), 3.53(t, J=8.0, 1H), 2.77 (t, J=6.4, 4H), 2.30-2.19 (m, 1H), 2.05 (q, J=6.8,8H), 1.88-1.75 (m, 2H), 1.69-1.51 (m, 4H), 1.42-1.19 (m, 36H), 0.89 (t,J=6.8, 6H). Calc. mass for C₄₁H₇₄O₃ is 614.5; found 637.3 (+Na).

Synthesis of Compound 7201

To a solution of compound 7013 (11.6 g, 18.9 mmol, 1.0 eq) and triethylamine (5.45 mL, 37.7 mmol, 2.0 eq) in dichloromethane at 0° C. was addeddrop wise a solution of methanesulfonyl chloride (1.74 mL, 22.67 mmol,1.2 eq), and the reaction was continued at room temperature for 1 h.After completion of the reaction, was washed with water, brine, andcombined organics were dried on MgSO₄. The concentrated mixture waspurified on column chromatography using hexane and ethyl acetate (20%)as gradients to get pure mesylated derivative (7201) as an oil in 93%yield. ¹H NMR (400 MHz, CDCl₃) δ 5.48-5.22 (m, 8H), 4.35 (qd, J=10.0,4.9, 2H), 4.25-4.14 (m, 1H), 4.13-4.03 (m, 1H), 3.53 (t, J=7.6, 1H),3.02 (s, 3H), 2.77 (t, J=6.4, 4H), 2.13-1.85 (m, 10H), 1.57 (dd, J=18.2,9.2, 4H), 1.44-1.15 (m, 36H), 0.89 (t, J=6.7, 6H). Calc. mass forC₄₂H₇₆O₅S is 693.1; found 693.2.

Synthesis of Compound 7202

To a solution of the compound 7201 (2.0 g, 3.0 mmol, 1.0 eq) in DMF wasadded solid NaN₃ (0.98 g, 15.0 mmol, 5.0 eq) at room temperature and thereaction was continued at 65° C. until the completion of the reaction.Reaction mixture was poured onto ice water, extracted into ethylacetate, combined organics were dried on Na₂SO₄, concentrated, purifiedon column chromatography using hexane and ethyl acetate (5%) asgradients to get pure azido (7202) derivative in 89% yield. ¹H NMR (400MHz, CDCl₃) δ 5.53-5.19 (m, 8H), 4.21-3.97 (m, 2H), 3.57-3.29 (m, 3H),2.76 (t, J=6.4, 4H), 2.04 (q, J=6.8, 8H), 1.80 (m, 2H), 1.66-1.43 (m,4H), 1.40-1.07 (m, 36H), 0.88 (t, J=6.8, 6H). Calc. mass for C₄₁H₇₃N₃O₂is 640.0; found 612.5 (—N₂).

Synthesis of Compound 7203

To a solution of compound 7202 (1.7 g, 2.65 mmol, 1.0 eq) in anhydroustetrahydrofuran, was added drop wise a 1M solution of LAH (3.98 mL, 3.98mmol, 1.5 eq) at 0° C. Reaction was continued at room temperature, aftercompletion of the reaction was quenched with saturated solution ofNa₂SO₄ slowly at 0° C. Compound was extracted into excess amount ofethyl acetate, organic layer was washed with brine, dried over Na₂SO₄,concentrated and further dried on vacuum to get pure amine (7203) in 90%yield, and this has been used directly without further purification. ¹HNMR (400 MHz, CDCl₃) δ 5.51-5.16 (m, 8H), 4.13 (dd, J=9.3, 3.6, 1H),4.03 (dd, J=7.5, 6.1, 1H), 3.46 (t, J=7.8, 1H), 2.96-2.67 (m, 6H),2.20-1.92 (m, 8H), 1.82-1.49 (m, 6H), 1.46-1.12 (m, 38H), 0.88 (t,J=6.8, 6H). Calc. mass for the C₄₁H₇₅NO₂ is 614.0; found 614.5.

Synthesis of Compound 7204 (ALNY-232)

To a solution of amine 7203 (0.61 g, 1.0 mmol, 1.0 eq) and DIPEA (1.84mL, 10.0 mmol, 10.0 eq) in a solvent mixture (DCM:DMF) was added1H-pyrazole-1-carboxamidine hydrochloride (1.46 g, 10.0 mmol, 10.0 eq)portion wise at room temperature, under argon atmosphere. The reactionwas continued for overnight, after completion of the reaction, waspoured onto ice, and extracted with the ethyl acetate. The combinedorganics were washed with water, brine, dried over Na₂SO₄ and purifiedby preparative chromatography to get pure 0.16 g (25%) of the guanidinederivative (7204). 1H NMR (400 MHz, CDCl3) δ 11.76 (s, 1H), 7.99 (t,J=6.3, 1H), 7.44 (s, 2H), 5.48-5.20 (m, 8H), 4.24-4.00 (m, 2H), 3.54(dd, J=7.3, 6.2, 1H), 3.32 (d, J=3.0, 2H), 3.09 (dt, J=10.5, 5.3, 1H),2.76 (t, J=6.5, 4H), 2.03 (q, J=6.8, 8H), 1.90-1.77 (m, 1H), 1.76-1.49(m, 6H), 1.48-1.05 (m, 34H), 0.87 (dd, J=6.8, 6H). ¹³C NMR (101 MHz,cdcl₃) δ 158.96, 130.41, 130.36, 130.33, 128.18, 128.14, 113.52, 77.54,77.22, 76.90, 76.60, 72.36, 69.54, 46.09, 38.39, 37.68, 37.01, 34.09,31.74, 30.10, 29.92, 29.78, 29.76, 29.56, 29.55, 29.53, 27.47, 27.46,27.41, 25.84, 24.37, 24.12, 22.79, 14.31, 8.86. Calc. mass for theC₄₂H₇₇N₃O₂ is 656.0; found 656.2.

Example 17: Synthesis of Ester Linked Lipids

Ester Analogs

Experimental Compound 7002

Magnesium (711 mg, 29.25 mmol) was placed in a round bottle flask. THF(30 mL) and 2-3 mg of I₂ were added. The mixture was warmed at 50° C.and oleylbromide (7001, 6.46 g, 19.50 mmol) was added slowly. When ˜1 mLof oleylbromide was added, formation of the Grignard reagent wasinitiated. After addition of the rest of oleylbromide, the Grignardreagent was stirred at room temperature for 60 min then slowly added toa solution of 1,1′-carbonyldiimidazole (1.54 g, 9.51 mmol) in THF (100mL) at −50° C. The reaction mixture was kept stirring at −50° C. for 30min then at room temperature for 60 min. The reaction was quenched with40 mL of saturated NH₄Cl aq. and the mixture was extracted with Et₂O andH₂O. The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% Et₂O inHexane) to give compound 7002 (2.70 g, 5.09 mmol, 53%, R_(f)=0.48developed with 5% EtOAc in Hexane). Molecular weight for C₃₇H₇₁O (M+H)⁺Calc. 531.55. Found 531.5.

Compound 7003

To a solution of compound 7002 (1.36 g, 2.56 mmol) in THF (25 mL), 1 Mlithium aluminum hydride in THF (5.12 mL, 5.12 mmol) was added at 0° C.The reaction mixture was stirred at room temperature for 3 hours. Thereaction was quenched with saturated Na₂SO₄ aq. (20 mL), then extractedwith Et₂O and H₂O. The organic layer was dried over MgSO₄, filtered andconcentrated. The crude was purified by silica gel column chromatography(0-5% Et₂O in Hexane) to give compound 7003 (942 mg, 1.77 mmol, 69%,R_(f)=0.26 developed with 5% EtOAc in Hexane).

Compound 7004

To a solution of compound 7003 (940 mg, 1.76 mmol) and4-(dimethylamino)butyric acid hydrochloride (355 mg, 2.12 mmol) inCH₂Cl₂ (15 mL), diisopropylethylamine (0.920 mL, 5.28 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (406 mg,2.12 mmol) and DMAP (43 mg, 0.352 mmol) were added. The reaction mixturewas stirred at room temperature for 14 hours. The reaction mixture wasdiluted with CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ aq. (50mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 7004 (817 mg, 1.26 mmol, 72%, R_(f)=0.29developed with 5% MeOH in CH₂Cl₂). Molecular weight for C₄₃H₈₄NO₂ (M+H)⁺Calc. 646.65. Found 646.5.

Compound 7017

To a stirred solution of ketone 7016 (1.5 g, 2.84 mmol, 1.0 eq) inmethanol and THF (2:1) was added solid NaBH₄ (0.16 g, 4.26 mmol, 1.5 eq)at 0° C. portions wise and continued the reaction at room temperatureuntil completion of the reaction. Reaction was quenched with drop wiseaddition of 2N HCl solution at ice cold temperature, organic solvent wasevaporated and re-dissolved in ethyl acetate, washed with water, brine,combined organics were dried over MgSO₄, concentrated and purified bycolumn chromatography using hexane: ethyl acetate (20%) as gradients toget pure alcohol 7017 in 94% (1.42 g) yields. ¹H NMR (400 MHz, CDCl₃) δ5.49-5.20 (m, 6H), 3.57 (s, 1H), 2.76 (t, J=6.4, 2H), 2.13-1.88 (m, 9H),1.51-1.12 (m, 53H), 0.95-0.75 (m, 6H). Calc. mass for the C₃₇H₇₀O:530.5; found 531.5.

Compound 7018

Prepared by similar experimental conditions used as for compound 7010,using alcohol 7017 (1.42 g, 2.68 mmol, 1.0 eq), N,N-dimethylaminobutyric acid hydrochloride (0.53 g, 3.21 mmol, 1.2 eq), DIPEA (1.48 mL,8.0 mmol, 3.0 eq), EDCI (0.56 g, 2.94 mmol, 1.1 eq), DMAP (0.065 g, 0.53mmol, 0.1 eq) in DCM gave 1.34 g (78%) of the pure product 7018. ¹H NMR(400 MHz, CDCl₃) δ 5.47-5.20 (m, 8H), 4.92-4.77 (m, 1H), 2.76 (t, J=6.3,2H), 2.28 (dt, J=16.6, 7.5, 411), 2.20 (s, 6H), 2.08-1.89 (m, 8H),1.83-1.70 (m, 2H), 1.48 (d, J=5.2, 4H), 1.38-1.16 (m, 40H), 0.91-0.80(m, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 173.61, 130.51, 130.40, 130.35,130.13, 130.05, 128.16, 128.13, 77.55, 77.23, 76.91, 74.46, 59.18,45.68, 34.36, 32.84, 32.69, 32.13, 31.75, 29.99, 29.92, 29.89, 29.78,29.76, 29.72, 29.67, 29.58, 29.54, 29.52, 29.40, 29.36, 27.45, 27.43,25.84, 25.57, 23.39, 22.91, 22.80, 14.36, 14.31. Calc. mass for theC43H81NO2: 643.6; found 644.5.

Compound 7020

Prepared by similar experimental conditions used as compound 7017, usingketone 57 (0.75 g, 1.43 mmol, 1.0 eq) in methanol and THF (2:1) wasadded solid NaBH₄ (0.08 g, 2.14 mmol, 1.5 eq) in methanol: THF, gave0.63 g (84%) of the pure alcohol 7020. ¹H NMR (400 MHz, CDCl₃) δ5.48-5.20 (m, 10H), 3.57 (s, 1H), 2.88-2.65 (m, 6H), 2.06 (dq, J=14.0,7.1, 8H), 1.50-1.18 (m, 35H), 0.96 (t, J=7.5, 3H), 0.88 (dd, J=12.8,6.2, 3H). Calc. mass for the C₃₇H₆₆O: 526.5; found 527.5.

Preparation of Compound 7021

Prepared by similar experimental conditions used as for compound 7010,using alcohol 7020 (0.62 g, 1.18 mmol, 1.0 eq), N,N-dimethylaminobutyric acid hydrochloride (0.23 g, 1.41 mmol, 1.2 eq), DIPEA (0.65 mL,3.54 mmol, 3.0 eq), EDCI (0.24 g, 1.3 mmol, 1.1 eq), DMAP (0.028 g, 0.23mmol, 0.1 eq) in DCM gave 0.63 g (84%) of the pure product 7021. ¹H NMR(400 MHz, CDCl₃) δ 5.46-5.19 (m, 8H), 4.91-4.78 (m, 1H), 2.85-2.68 (m,6H), 2.29 (dt, J=15.2, 7.5, 4H), 2.20 (s, 6H), 2.11-1.95 (m, 8H), 1.78(dd, J=14.8, 7.5, 2H), 1.49 (d, J=5.5, 4H), 1.40-1.17 (m, 32H), 0.96 (t,J=7.5, 3H), 0.87 (t, J=6.8, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.17,126.73, 125.15, 124.98, 124.93, 123.06, 123.04, 122.75, 122.72, 122.43,121.91, 72.13, 71.81, 71.49, 69.04, 53.75, 40.25, 28.95, 27.27, 26.33,24.47, 24.45, 24.36, 24.33, 24.29, 24.15, 24.10, 22.05, 22.04, 22.00,20.42, 20.41, 20.32, 20.15, 17.96, 17.38, 15.35, 9.08, 8.88. Calc. massfor the C₄₃H₇₇NO₂: 639.6; found 640.5.

Compound 7023

To a solution of compound 7022 in methanol:ethyalcetate solvent mixture(2:1) was added 10% Pd/C, removed air by vacuum, purged with argon,repeated the cycle (2×), finally purged with H₂, and continued thereaction under H₂ at room temperature for overnight. After completion ofthe reaction, filtered through small pad of celite, washed with ethylacetate, evaporated the solvent and purified by column chromatographyusing dichloromethane: methanol (5%) as gradients to get pure whitesolid form of compound 7023 in 64% (0.64 g) yields. ¹H NMR (400 MHz,CDCl₃) δ 5.37 (s, OH), 4.85 (p, J=6.2, 1H), 2.29 (dt, J=14.8, 7.5, 4H),2.21 (s, 6H), 1.84-1.71 (m, 2H), 1.49 (d, J=5.4, 4H), 1.36-1.13 (m,64H), 0.87 (t, J=6.8, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 173.58, 77.54,77.22, 76.91, 74.49, 59.17, 45.64, 34.36, 32.83, 32.70, 32.15, 29.92,29.88, 29.81, 29.78, 29.58, 25.55, 23.36, 22.91, 14.33. Calc. mass forthe C43H87NO2: 649.6; found 650.8.

Example 18: Ester Synthesis

DLin-M-C1-DMA

DLin-M-C1-DMA. A solution of dilinolenylmethanol (0.50 g),N,N-dimethylglycine (0.53 g), 4-N,N-dimethylaminopyridine (0.60 g) and1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.50 g) inmethylene chloride (5 mL) was stirred at room temperature. The reactionwas monitored by TLC. When all of the dilinolenylmethanol had beenconverted, the reaction mixture was washed with dilute hydrochloricacid, followed by dilute sodium bicarbonate solution. The organicfractions were dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel column using a0-3% methanol/methylene chloride elution gradient, yieldingDLin-M-C1-DMA (0.35 g) as a colorless oil.

¹HNMR: (CDCl₃) δ 0.91 (t; J=6.8 Hz; 6H); 2.07 (m; 8H); 2.42 (s; 6H);2.79 (t; J=6.5 Hz; 4H); 3.21 (s; 2H); 4.97 (m; 1H); 5.37 (m; 8H)

DLin-M-C4-DMA

N,N-dimethyl-5-aminopentanoic acid. Bromovaleric acid (2 g) wasdissolved in aqueous solution of dimethylamine and stirred at roomtemperature overnight. The solvent was removed on a rotovap and theresidue treated with an aqueous solution containing one equivalent ofsodium bicarbonate. The solvent was removed, the residue suspended inethanol and filtered. The solvent was removed from the filtrate and theresidue suspended in methylene chloride and suspended again. Afterfiltration, removal of the solvent from the filtrate yielded an oil (1.3g) that slowly crystallized on storage.

DLin-M-C4-DMA; as described for DLin-M-C1-DMA usingN,N-dimethyl-5-aminopentanoic acid.

¹HNMR: (CDCl₃) δ 0.91 (t; J=6.9 Hz; 6H); 1.67 (m; 2H); 2.07 (m; 8H);2.32 (s; 6H); 2.37 (m; 4H); 2.79 (t; J=6.5 Hz; 4H); 4.88 (m; 1H); 5.37(m; 8H)

DLin-M-05-DMA

N,N-dimethyl-6-aminobutanoic acid; as described forN,N-dimethyl-5-aminopentanoic acid using 6-bromobutanoic acid.

DLin-M-05-DMA; as described for DLin-M-C1-DMA usingN,N-dimethyl-6-aminobutanoic acid.

¹HNMR: (CDCl₃) δ 0.91 (t; J=6.9 Hz; 6H); 1.66 (m); 2.07 (m; 8H); 2.31(t; J=7.5 Hz; 2H); 2.39 (s; 6H); 2.47 (bm; 2H); 4.88 (m; 1H); 5.37 (m;8H)

DLen-K5-C2-DMA

Len-Br. A solution of linolenyl mesylate (2.2 g) and lithium bromide(2.5 g) in acetone (25 mL) was stirred at room temperature overnight.Methylene chloride was added and the solution washed twice with water.The organic fractions were dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel column using a 0-2% ethyl acetate/hexane elution gradient, yieldingLen-Br (2.1 g) as a colorless oil.

DLen-M-formate. A solution of Len-Br (2.1 g) in anhydrous diethyl ether(60 mL) was treated with magnesium filings (180 mg) under refluxovernight. The solution was allowed to cool and ethyl formate (0.5 mL)added dropwise. The reaction was stirred at room temperature for threehours. Aqueous sulfuric acid (5%, 40 mL) was added and the solutionextracted with diethyl ether. The organic fraction was washed withsaline, dried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel column using a 0-3%ethyl acetate/hexane elution gradient, yielding crude DLen-M-formate asa colorless oil.

DLen-M. Crude DLen-M-formate prepared above was treated with a 5%solution of sodium hydroxide in water/ethanol (10 mL, 10:90 v/v) for 30minutes. The solution was diluted with water and extracted withmethylene chloride. The organic fractions were dried over anhydrousmagnesium sulfate, filtered and the solvent removed. The residue waspassed down a silica gel column using a 0-10% ethyl acetate/hexaneelution gradient, yielding DLen-M as a colorless oil.

DLen-ketone. A solution of DLEN-M (prepared above) in methylene chloride(20 mL) was treated with pyridinium chlorochromate (1 g) at roomtemperature for two hours. Diethyl ether (50 mL) was added and theresultant suspension washed through a bed of silica gel (2×). Thesolvent was removed and the residue passed down a silica gel columnusing a 0-2% ethyl acetate/hexane gradient, yielding DLen-ketone (0.57g) as a colorless oil.

DLen-K5-C2-OH. A solution of DLen-ketone (0.57 g), pyridiniump-toluenesulfonate (0.10 g) and butan-1,2,4-triol (0.50 g) in toluene(100 mL) was refluxed in a Dean & Stark apparatus overnight. Thereaction mixture was partitioned between methylene chloride and brine.The organic fractions were dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel column using methylene chloride, yielding DLen-K5-C2-OH (0.52 g) asa colorless oil.

Procedure 09-028 (17 Apr. 9): DLen-K5-C2-OMs. A solution ofDLen-K5-C2-OH (0.52 g) in methylene chloride (20 mL) was treated withmethanesulfonyl anhydride (0.40 g) and triethylamine (0.7 mL) at roomtemperature overnight. The organic fraction was washed with saline,dried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was used in subsequent reactions without furtherpurification. DLen-K5-C2-DMA. A solution of crude DLen-K5-C2-OMs in 2.0M dimethylamine in THF (15 mL) was stirred at room temperature for twodays. The solvent was removed on a rotovap and the residue passed downsilica gel using a 0-6% methanol/methylene chloride gradient, yieldingDLen-K5-C2-DMA (0.34 g) as a colorless oil.

¹HNMR: (CDCl₃) δ 0.95 (t; J=7.5 Hz; 6H); 1.56 (m; 4H); 1.70 (m; 1H);1.81 (m; 1H); 2.05 (m; 8H); 2.27 (s; 6H); 2.36 (m; 1H); 2.46 (m; 1H);2.79 (t; J=6.0 Hz; 8H); 3.27 (t; J=7.2 Hz; 1H); 4.06 (m; 2H); 5.34 (m;12H)

DO-K5-C2-DMA

O-Br; as described for Len-Br using oleyl mesylate.

DO-M-formate; as described for DLen-M-formate using O-Br.

DO-M; as described for DLen-M using DO-M-formate.

DO-ketone; as described for DLen-ketone using DO-M.

DO-K5-C2-OH; as described for DLen-K5-C2-OH using DO-ketone.

DO-K5-C2-OMs; as described for DLen-K5-C2-OMs using DO-K5-C2-OH.

DO-K5-C2-DMA; as described for DLen-K5-C2-DMA using DO-K5-C2-OMs.

¹HNMR: (CDCl₃) δ 0.86 (t; J=6.8 Hz; 6H); 1.55 (m; 4H); 1.64 (m; 1H);1.79 (ddd; J=12.6 Hz, J′=11.2 Hz, J″=6.2 Hz; 1H); 1.99 (m; 8H); 2.20 (s;6H); 2.26 (ddd; J=12.2 Hz, J′=9.5 Hz; J″=5.9 Hz; 1H); 2.38 (ddd; J=11.9Hz, J′=9.7 Hz, J″=5.6 Hz; 1H); 3.46 (t; J=7.3 Hz; 1H); 4.05 (m; 2H);5.32 (m; 4H)

DLin-M-C3-A

Procedure 09-071 (14 Jul. 9): DLin-M-C3-A. A solution ofdilinolenylmethanol (0.51 g), N—BOC-4-aminobutyric acid (0.53 g),4-N,N-dimethylaminopyridine (0.39 g) and1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.30 g) inmethylene chloride (5 mL) was stirred at room temperature overnight. Thereaction mixture was washed with dilute hydrochloric acid. The organicfractions were dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was treated with triflouroacetic acid (2mL) at room temperature for an hour. The solution was diluted withmethylene chloride, washed with water and then washed with aqueoussodium bicarbonate. The organic fractions were dried over anhydrousmagnesium sulfate, filtered and the solvent removed. The residue waspassed down a silica gel column using a 0-10% methanol/methylenechloride elution gradient, yielding DLin-M-C3-A (0.45 g) as a colorlessoil.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.8 Hz; 6H); 1.75 (p; J=7.3 Hz; 2H); 2.03(m; 8H); 2.32 (t; J=7.4 Hz; 2H); 2.75 (m; 6H); 4.84 (p; J=6.2 Hz; 1H);5.35 (m; 8H)

DLin-M-C3-MA

DLin-M-C3-Br. A solution of dilinolenylmethanol (0.5 g) in methylenechloride (20 mL) was treated with 4-bromobutyryl chloride (1 mL) andtriethylamine (1 mL) with stirring at room temperature overnight. Thereaction mixture was diluted with water, acidified with hydrochloricacid and extracted with methylene chloride. The organic fractions weredried over anhydrous magnesium sulfate, filtered and the solventremoved. The crude DLin-M-C3-Br was used in subsequent reactions withoutfurther purification. Procedure 09-061 (16 Jun. 9): DLin-M-C3-MA. Asolution of DLin-M-C3-Br (0.51 g) was treated with a solution ofmethylamine in THF/methylene chloride (50 mL; 20/30 v/v) at roomtemperature. The reaction was monitored by TLC. When the reaction wascomplete the solvent was removed on a rotovap. The residue waspartitioned between methylene chloride and dilute hydrochloric acid. Theorganic phase was washed with dilute aqueous sodium bicarbonatesolution, dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel column using a0-4% methanol/methylene chloride elution gradient, yielding DLin-M-C3-MA(0.31 g) as a colorless oil.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.9 Hz; 6H); 1.82 (m; 2H); 2.03 (m; 8H);2.33 (t; J=7.4 Hz; 2H); 2.43 (s; 3H); 2.62 (t; J=7.1 Hz; 2H); 2.75 (t;J=6.4 Hz; 4H); 4.84 (p; J=6.3 Hz; 1H); 5.35 (m; 8H)

DLin-M-C3-EA

DLin-M-C3-EA; as described for DLin-M-C3-MA using ethylamine.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.8 Hz; 6H); 1.10 (t; J=7.1 Hz; 3H); 1.82(p; J=7.3 Hz; 2H); 2.03 (m; 8H); 2.33 (t; J=7.4 Hz; 2H); 2.65 (q; J=7.0Hz; 4H); 2.62 (t; J=7.1 Hz; 2H); 2.75 (t; J=6.4 Hz; 4H); 4.84 (p; J=6.3Hz; 1H); 5.33 (m; 8H)

DLin-M-C3-IPA

DLin-M-C3-IPA; as described for DLin-M-C3-MA using isopropylamine.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.8 Hz; 6H); 1.03 (d; J=6.2 Hz; 6H); 1.78(p; J=7.3 Hz; 2H); 2.03 (m; 8H); 2.32 (t; J=7.4 Hz; 2H); 2.60 (t; J=7.3Hz; 2H); 2.77 (m; 5H); 4.84 (p; J=6.2 Hz; 1H); 5.34 (m; 8H)

DLin-M-C3-DEA (ED50=0.3)

DLin-M-C3-DEA; as described for DLin-M-C3-MA using diethylamine.

DLin-M-C3-DIPA (ED50=4.5)

DLin-M-C3-DIPA; as described for DLin-M-C3-MA using diisopropylamine.

DLin-M-C3-MIPA

DLin-M-C3-MIPA; as described for DLin-M-C3-MA usingmethylisopropylamine.

DLin-M-C3-EIPA

DLin-M-C3-EIPA; as described for DLin-M-C3-MA using ethylisopropylamine.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.8 Hz; 6H); 0.94 (d; J=6.2 Hz; 6H); 0.99(t; J=7.1 Hz; 3H); 1.71 (m; 2H); 2.03 (m; 8H); 2.30 (t; J=7.3 Hz; 2H);2.37 (m; 2H); 2.43 (q; J=7.1 Hz; 2H); 2.75 (t; J=6.4 Hz; 4H); 2.90 (m;1H); 4.84 (m; 1H); 5.34 (m; 8H)

DLin-M-C3-MEA

DLin-M-C3-MEA; as described for DLin-M-C3-MA using methylethylamine.

¹HNMR: (CDCl₃) δ 0.87 (t; J=6.9 Hz; 6H); 1.02 (t; J=7.2 Hz; 3H); 1.77(m; 2H); 2.03 (m; 8H); 2.19 (s; 3H); 2.30 (m; 4H); 2.39 (q; J=7.2 Hz;2H); 2.75 (t; J=6.5 Hz; 4H); 4.84 (m; 111); 5.34 (m; 8H)

Example 19: Synthesis of2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6S-C1-DMA)

1. Synthesis of Linoleyl Bromide (II)

A mixture of linoleyl methane sulfonate (26.6 g, 77.2 mmol) and lithiumbromide (30.5 g, 350 mmol) in acetone (350 mL) was stirred undernitrogen for two days. The resulting suspension was filtered and thesolid washed with acetone. The filtrate and wash were combined andsolvent evaporated. The resulting residual was treated with water (300mL). The aqueous phase was extracted with ether (3×150 mL) The combinedether phase was washed with water (200 mL), brine (200 mL) and driedover anhydrous Na₂SO₄. The solvent was evaporated to afford 29.8 g ofyellowish oil. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 700 mL) eluted with hexanes. This gave 20.8g (82%) of linoleyl bromide (II).

2. Synthesis of Dilinoleylmethyl Formate (III)

To a suspension of Mg turnings (1.64 g, 67.4 mmol) with one crystal ofiodine in 500 mL of anhydrous ether under nitrogen was added a solutionof linoleyl bromide (II, 18.5 g, 56.1 mmol) in 250 mL of anhydrous etherat room temperature. The resulting mixture was refluxed under nitrogenovernight. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added dropwise ethyl formate (4.24 g, 57.2mmol). Upon addition, the mixture was stirred at room temperatureovernight. The mixture was treated with 10% H₂SO₄ aqueous solution (250mL). The ether phase was separated and aqueous phase extracted withether (150 mL). The combined organic phase was washed with water (400mL), brine (300 mL), and then dried over anhydrous Na₂SO₄. Evaporationof the solvent gave 17.8 g of yellowish oil as a crude product (III).The crude product was used directly in the following step withoutfurther purification.

3. Synthesis of Dilinoleyl Methanol (IV)

The above crude dilinoleylmethyl formate (III, 17.8 g) and KOH (3.75 g)were stirred in 85% EtOH at room temperature under nitrogen overnight.Upon completion of the reaction, most of the solvent was evaporated. Theresulting mixture was poured into 150 mL of 5% HCl solution. The aqueousphase was extracted with ether (2×150 mL). The combined ether extractwas washed with water (2×100 mL), brine (100 mL), and dried overanhydrous Na₂SO₄. Evaporation of the solvent gave 20.0 of dilinoleylmethanol (IV) as yellowish oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 700 mL) eluted with 0-5%ethyl acetate gradient in hexanes. This gave 9.6 g of dilinoleylmethanol (IV).

4. Synthesis of Dilinoleyl Ketone (V)

To a mixture of dilinoleyl methanol (4.0 g, 7.2 mmol) and anhydrouspotassium carbonate (0.4 g) in 100 mL of CH₂Cl₂ was added pyridiniumchlorochromate (PCC, 4.0 g, 19 mmol). The resulting suspension wasstirred at room temperature for 2 hours. Ether (300 mL) was then addedinto the mixture, and the resulting brown suspension was filteredthrough a pad of silica gel (150 mL). The silica gel pad was furtherwashed with ether (3×75 mL). The ether filtrate and washes werecombined. Evaporation of the solvent gave 5.1 g of an oily residual as acrude product. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 200 mL) eluted with 0-4% ethyl acetate inhexanes. This afforded 3.0 g (79%) of dilinoleyl ketone (V).

5. Synthesis of 2,2-Dilinoleyl-5-hydroxymethyl)-[1,3]-dioxane (VI)

A mixture of dilinoleyl ketone (V, 1.05 g, 2.0 mmol),2-hydroxymethyl-1,3-propanediol (490 mg, 4.2 mmol) and pyridiniump-toluenesulfonate (100 mg, 0.4 mmol) in 150 mL of toluene was refluxedunder nitrogen overnight with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×100 mL), brine (100 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (1.2 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 100 mL) with 0-5% methanol gradient in dichloromethane aseluent. This afforded 0.93 g of pure VI as pale oil.

6. Synthesis of 2,2-Dilinoleyl-5-methanesulfonylmethyl-[1,3]-dioxane(VII)

To a solution of 2,2-dilinoleyl-5-hydroxymethyl-[1,3]-dioxane (VI, 0.93g, 1.5 mmol) and dry triethylamine (290 mg, 2.9 mmol) in 50 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (400 mg, 2.3 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×75 mL), brine (75mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 1.0 g of pale oil. The crude product was used in the followingstep without further purification.

7. Synthesis of 2,2-Dilinoleyl-5-dimethylaminomethyl)-[1,3]-dioxane(DLin-K6S-C1-DMA)

To the above crude material (VII, 1.0 g) under nitrogen was added 20 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 7 days. An oily residual was obtained uponevaporation of the solvent. Column chromatography on silica gel (230-400mesh, 100 mL) with 0-3% methanol gradient in chloroform as eluentresulted in 150 mg of the product DLin-K6S-C1-DMA as pale oil. ¹H NMR(400 MHz, CDCl₃) δ: 5.24-5.51 (8, m, 4×CH═CH), 4.04 (2H, dd, 2×OCH)),3.75 (2H, dd OCH), 2.7-2.9 (2H, br, NCH₂), 2.78 (4H, t, 2×C═C—CH₂—C═C),2.57 (6H, s, 2×NCH₃), 1.95-2.17 (9H, q, 4×allylic CH₂ and CH), 1.67-1.95(2H, m, CH₂), 1.54-1.65 (4H, m, 2×CH₂), 1.22-1.45 (32H, m), 0.90 (6H, t,2×CH₃) ppm.

Example 20: Synthesis of2,2-Dilinoleyl-5-dimethylaminobutyl-[1,3]-dioxane (DLin-K6S-C4-DMA)

This compound was synthesized as pale oil in a similar manner to that inExample 19, where 2-hydroxymethyl-1,3-propanediol was replaced with2-hydroxybutyl-1,3-propanediol. ¹H NMR (400 MHz, CDCl₃) δ: 5.24-5.45 (8,m, 4×CH═CH), 3.79 (2H, dd, 2×OCH)), 3.50 (2H, dd OCH), 2.76 (4H, t,2×C═C—CH₂—C═C), 2.37 (2H, t, NCH₂), 2.31 (6H, s, 2×NCH₃), 2.04 (8H, q,4×allylic CH₂), 1.63-1.90 (3H, m,), 1.45-1.62 (4H, m, 2×CH₂), 1.22-1.45(36H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 21: Synthesis of2,2-Dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxane (DLin-K6S-C2-DMA)

This compound was synthesized as pale oil in a similar manner to that inExample 19, where 2-hydroxymethyl-1,3-propanediol was replaced with2-hydroxyethyl-1,3-propanediol. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8,m, 4×CH═CH), 3.87 (2H, dd, 2×OCH)), 3.55 (2H, dd OCH), 2.75 (4H, t,2×C═C—CH₂—C═C), 2.45-2.60 (2H, br, NCH₂), 2.40 (6H, s, 2×NCH₃), 2.03(8H, q, 4×allylic CH₂), 1.73-1.86 (1H, m), 1.56-1.72 (6H, m, 2×CH₂),1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 22: Synthesis of2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane (DLin-K6A-C2-DMA)

1. Synthesis of 1,3,5-Pentanetriol (II)

Diethyl 3-hydroxyglutarate (I, 1.0 g, 4.9 mmol) in anhydrous THF (10 mL)was added dropwise to a suspension of LiAlH₄ in anhydrous THF (110 mL)under nitrogen with a cold water bath. Upon addition, the bath wasremoved and the suspension was stirred at room temperature for 2 days.The resulting mixture was quenched by adding 13 mL of brine very slowlywith an ice-water bath. A white suspension was resulted, and the mixturewas stirred at room temperature overnight. The solid was filtered, andwashed with THF. The filtrate and wash were combined, and solventevaporated to give 0.70 g of pale oil. Column chromatography of thecrude product (230-400 mesh SiO₂, 100 mL, 0-12% methanol gradient inchloroform) afforded 0.54 g of II as colourless oil.

2. Synthesis of 2,2-Dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxane (IV)

A mixture of dilinoleyl ketone (III, 0.80 g, 1.5 mmol),1,3,5-pentanetriol (II, 0.54 g, 4.5 mmol) and pyridiniump-toluenesulfonate (60 mg, 0.24 mmol) in 150 mL of toluene was refluxedunder nitrogen overnight with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×75 mL), brine (75 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (1.1 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 75 mL) with 0-3% methanol gradient in dichloromethane aseluent. This afforded 0.75 g (79%) of pure IV as colourless oil.

3. Synthesis of 2,2-Dilinoleyl-4-(2-methanesulfonylethyl)-[1,3]-dioxane(V)

To a solution of 2,2-dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxane (IV,0.75 g, 1.2 mmol) and dry triethylamine (0.58 g, 5.7 mmol) in 40 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.50 g, 2.9 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×50 mL), brine (50mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 0.80 g of pale oil as a crude product. The crude product was usedin the following step without further purification.

4. Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane(DLin-K6A-C2-DMA)

To the above crude material (V, 0.80 g) under nitrogen was added 15 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 6 days. The solid was filtered. An oily residualwas obtained upon evaporation of the solvent. Column chromatography onsilica gel (230-400 mesh, 100 mL) with 0-6% methanol gradient indichloromethane as eluent resulted in 0.70 g of the productDLin-K6A-C2-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.28-5.45 (8, m,4×CH═CH), 3.85-4.0 (2H, m, 2×OCH), 3.78 (1H, dd, OCH), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.55-2.90 (2H, br, NCH₂), 2.47 (6H, s, 2×NCH₃), 2.05(8H, q, 4×allylic CH₂), 1.65-1.90 (4H, m, CH₂), 1.47-1.65 (4H, m, CH₂),1.1-1.65 (36H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 23: Synthesis of2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxane (DLin-K6A-C3-DMA)

1. Synthesis of 1,3,6-Hexanetriol (II)

Diethyl β-ketoadipate (I, 1.86 g, 8.6 mmol) was added dropwise to asuspension of LiAlH₄ in anhydrous THF (90 mL) under argon with anice-water bath. Upon addition, the bath was removed and the suspensionwas stirred at room temperature overnight. The resulting mixture wasquenched by adding 10 mL of brine very slowly with an ice-water bath. Awhite suspension was resulted, and the mixture was stirred at roomtemperature overnight. The solid was filtered, and washed with THFfollowed by EtOH (2×50 mL). The filtrate and wash were combined, andsolvent evaporated to give 0.90 g of pale oil. Column chromatography ofthe crude product (230-400 mesh SiO₂, 100 mL, 0-10% methanol gradient indichloromethane) afforded 0.70 g of II as colourless oil.

2. Synthesis of 2,2-Dilinoleyl-4-(3-hydroxypropyl)-[1,3]-dioxane (IV)

A mixture of dilinoleyl ketone (III, 1.80 g, 3.4 mmol),1,3,6-hexanetriol (II, 0.50 g, 3.7 mmol) and pyridiniump-toluenesulfonate (100 mg, 0.40 mmol) in 120 mL of toluene was refluxedunder argon for 3 hours with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×50 mL), brine (50 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (2.0 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 50 mL) with 0-3% methanol gradient in dichloromethane aseluent. This afforded 0.90 g (41%) of pure IV as colourless oil.

3. Synthesis of 2,2-Dilinoleyl-4-(3-methanesulfonylpropyl)-[1,3]-dioxane(V)

To a solution of 2,2-dilinoleyl-4-(3-hydroxypropyl)-[1,3]-dioxane (IV,0.97 g, 1.5 mmol) and dry triethylamine (0.44 g, 4.3 mmol) in 60 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.60 g, 3.5 mmol)under argon. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×30 mL), brine (30mL), and dried over anhydrous MgSO₄. The solvent was evaporated toafford 1.1 g of pale oil as a crude product. The crude product was usedin the following step without further purification.

4. Synthesis of 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxane(DLin-K6A-C3-DMA)

To the above crude material (V, 1.1 g) under argon was added 20 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 5 days. The solid was filtered. An oily residual wasobtained upon evaporation of the solvent. Column chromatography onsilica gel (230-400 mesh, 40 mL) with 0-7% methanol gradient indichloromethane as eluent resulted in 0.85 g of the productDLin-K6A-C3-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8, m,4×CH═CH), 3.7-4.0 (3H, m, 3×OCH), 2.77 (4H, t, 2×C═C—CH₂—C═C), 2.5-2.8(2H, br, NCH₂), 2.5 (6H, s, 2×NCH₃), 2.05 (8H, q, 4×allylic CH₂),1.65-1.90 (4H, m, 2×CH₂), 1.40-1.65 (4H, m, 2×CH₂), 1.1-1.65 (38H, m),0.90 (6H, t, 2×CH₃) ppm.

Example 24: Synthesis of2,2-Diarachidonyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DAra-K5-C2-DMA)

1. Synthesis of Arachidonyl Bromide (II)

A mixture of arachidonyl methane sulfonate (1.0 g, 2.7 mmol) andmagnesium bromide (2.2 g, 12 mmol) in anhydrous ether (40 mL) wasstirred under argon for two days. The resulting suspension was filteredand the solid washed with ether (2×10 mL). The filtrate and wash werecombined and solvent evaporated. The resulting residual was treated withhexanes (50 mL). The solid was filtered and solvent evaporated resultingin an oily residual. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 30 mL) eluted with hexanes.This gave 1 g of arachidonyl bromide (II) as colourless oil.

2. Synthesis of Diarachidonylmethyl Formate (III)

To a solution of arachidonyl bromide (II, 1 g, 3 mmol) in anhydrousether (30 mL) was added Mg turnings (78 mg, 3.2 mmol) followed by onecrystal of iodine. The resulting mixture was refluxed under nitrogen for10 hours. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added ethyl formate (0.25 mL), and theresulting mixture was stirred at room temperature overnight. To themixture was added 20 mL of 10% H₂SO₄ aqueous solution. The ether phasewas separated and aqueous phase extracted with ether (30 mL). Thecombined organic phase was washed with water (2×25 mL), brine (25 mL),and then dried over anhydrous Na₂SO₄. Evaporation of the solvent gave1.1 g of pale oil as a crude product (III). The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)eluted with 0-3% ethyl acetate gradient in hexanes. This afforded 0.43 g(40%) of diarachidonylmethyl formate (III) as pale oil.

3. Synthesis of Diarachidonyl Methanol (IV)

The above diarachidonylmethyl formate (III, 0.43 g, 0.71 mmol) and KOH(100 mg) were stirred in 95% EtOH (20 mL) at room temperature undernitrogen overnight. Upon completion of the reaction, most of the solventwas evaporated. The resulting mixture was treated with 20 mL of 2M HClsolution. The aqueous phase was extracted with ether (2×30 mL). Thecombined ether extract was washed with water (2×25 mL), brine (25 mL),and dried over anhydrous Na₂SO₄. Evaporation of the solvent gave 0.44 gof IV as pale oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 40 mL) eluted with 0-5%ethyl acetate gradient in hexanes. This gave 0.41 g of diarachidonylmethanol (IV) as colourless oil.

4. Synthesis of Diarachidonyl Ketone (V)

To a mixture of diarachidonyl methanol (IV, 0.41 g, 0.71 mmol) andanhydrous potassium carbonate (0.05 g) in 10 mL of CH₂Cl₂ was addedpyridinium chlorochromate (PCC, 0.50 g, 2.3 mmol). The resultingsuspension was stirred at room temperature for 90 mins. Ether (50 mL)was then added into the mixture, and the resulting brown suspension wasfiltered through a pad of floresil (30 mL). The pad was further washedwith ether (3×30 mL) The ether filtrate and washes were combined.Evaporation of the solvent gave 0.40 g of an oily residual as a crudeproduct. The crude product was purified by column chromatography onsilica gel (230-400 mesh, 10 mL) eluted with 0-3% ether in hexanes. Thisafforded 0.30 g (75%) of diarachidonyl ketone (V). ¹H NMR (400 MHz,CDCl₃) δ: 5.3-5.5 (16H, m, 8×CH═CH), 2.82 (12H, t, 6×C═C—CH₂—C═C), 2.40(4H, t, 2×CO—CH₂), 2.08 (8H, m, 4×allylic CH₂), 1.25-1.65 (20H, m), 0.90(6H, t, 2×CH₃) ppm.

5. Synthesis of 2,2-Diarachidonyl-4-(2-hydroxyethyl)[1,3]-dioxolane (VI)

A mixture of diarachidonyl ketone (V, 0.30 g, 0.52 mmol),1,2,4-butanetriol (0.25 g, 2.4 mmol) and pyridinium p-toluenesulfonate(20 mg) in 60 mL of toluene was refluxed under argon overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (30 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)with 0-2% methanol in dichloromethane as eluent. This afforded 0.29 g(84%) of pure VI as pale oil.

6. Synthesis of2,2-Diarachidonyl-4-(2-methanesulfonylethyl)[1,3]-dioxolane (VII)

To a solution of 2,2-diarachidonyl-4-(2-hydroxyethyl)[1,3]-dioxolane(VI, 0.29 g, 0.43 mmol) and dry triethylamine (254 mg, 2.5 mmol) in 20mL of anhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.20 g, 1.1mmol) under nitrogen. The resulting mixture was stirred at roomtemperature overnight. The mixture was diluted with 30 mL of CH₂Cl₂. Theorganic phase was washed with water (2×25 mL), brine (25 mL), and driedover anhydrous MgSO₄. The solvent was evaporated to afford 0.30 g ofpale oil. The crude product was used in the following step withoutfurther purification.

7. Synthesis of2,2-Diarachidonyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DAra-K5-C2-DMA)

To the above crude material (VII, 0.30 g) under argon was added 15 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 40mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in0.18 g of the product DAra-K5-C2-DMA as pale oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.3-5.5 (16H, m, 8×CH═CH), 4.0-4.17 (2H, m, 2×OCH), 3.49 (1H,t, OCH), 2.65-2.85 (14H, m, 6×C═C—CH₂—C═C, NCH₂), 2.55 (6H, s, br,2×NCH₃), 2.06 (8H, m, 4×allylic CH₂), 1.80-1.92 (2H, m, CH₂), 1.4-1.75(4H, m, 2×CH₂), 1.22-1.45 (20H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 25: Synthesis of2,2-Didocosahexaenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DDha-K5-C2-DMA)

1. Synthesis of Docosahexaenoyl Bromide (II)

A mixture of docosahexaenoyl methane sulfonate (2.0 g, 5.1 mmol) andmagnesium bromide (4.3 g, 23 mmol) in anhydrous ether (100 mL) wasstirred under argon overnight. The resulting suspension was filtered andthe solid washed with ether (2×30 mL). The filtrate and wash werecombined and solvent evaporated. The resulting residual was purified bycolumn chromatography on silica gel (230-400 mesh, 40 mL) eluted withhexanes. This gave 2.2 g of docosahexaenoyl bromide (II) as colourlessoil.

2. Synthesis of Didocosahexaenoylmethyl Formate (III)

To a solution of docosahexaenoyl bromide (II, 2.2 g, 6.0 mmol) inanhydrous ether (60 mL) was added Mg turnings (145 mg, 6.0 mmol)followed by one crystal of iodine. The resulting mixture was refluxedunder argon for 5 hours. The mixture was cooled to room temperature. Tothe cloudy mixture under argon was added ethyl formate (0.50 mL), andthe resulting mixture was stirred at room temperature overnight. To themixture was added 40 mL of 5% H₂SO₄ aqueous solution. The ether phasewas separated and aqueous phase extracted with ether (50 mL). Thecombined organic phase was washed with water (2×50 mL), brine (50 mL),and then dried over anhydrous MgSO₄. Evaporation of the solvent gave 2.3g of yellowish oil as a crude product (III). The crude product waspurified by column chromatography on silica gel (230-400 mesh, 50 mL)eluted with 0-7% ethyl acetate gradient in hexanes. This afforded 1.38 g(65%) of didocosahexaenoylmethyl formate (III) as pale oil.

3. Synthesis of Didocosahexaenoyl Methanol (IV)

The above didocosahexaenoylmethyl formate (III, 1.38 g, 2.1 mmol) andKOH (300 mg) were stirred in 90% EtOH (70 mL) at room temperature undernitrogen for 90 min. Upon completion of the reaction, most of thesolvent was evaporated. The resulting mixture was treated with 60 mL of2M HCl solution. The aqueous phase was extracted with ether (2×75 mL).The combined ether extract was washed with water (2×50 mL), brine (50mL), and dried over anhydrous MgSO₄. Evaporation of the solvent gave1.18 g of crude IV as yellowish oil. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with0-6% ethyl acetate gradient in hexanes. This gave 1.0 g ofdidocosahexaenoyl methanol (IV) as colourless oil.

4. Synthesis of Didocosahexaenoyl Ketone (V)

To a mixture of didocosahexaenoyl methanol (IV, 1.2 g, 1.9 mmol) andanhydrous potassium carbonate (0.1 g) in 30 mL of CH₂Cl₂ was addedpyridinium chlorochromate (PCC, 1.05 g, 4.8 mmol). The resultingsuspension was stirred at room temperature for 2 hours. Ether (120 mL)was then added into the mixture, and the resulting brown suspension wasfiltered through a pad of silica gel (75 mL). The pad was further washedwith ether (3×75 mL) The ether filtrate and washes were combined.Evaporation of the solvent gave 1.3 g of an oily residual as a crudeproduct. The crude product was purified by column chromatography onsilica gel (230-400 mesh, 40 mL) eluted with 0-3% ethyl acetate inhexanes. This afforded 0.83 g (69%) of didocosahexaenoyl ketone (V).

5. Synthesis of 2,2-Didocosahexaenoyl-4-(2-hydroxyethyl)-[1,3]-dioxolane(VI)

A mixture of diarachidonyl ketone (V, 0.43 g, 0.69 mmol),1,2,4-butanetriol (0.35 g, 3.3 mmol) and pyridinium p-toluenesulfonate(50 mg) in 75 mL of toluene was refluxed under argon overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (30 mL), and dried over anhydrous MgSO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)with 0-2% methanol in dichloromethane as eluent. This afforded 0.43 g(95%) of pure VI as pale oil.

6. Synthesis of2,2-Didocosahexaenoyl-4-(2-methanesulfonylethyl)[1,3]-dioxolane (VII)

To a solution of2,2-didocosahexaenoyl-4-(2-hydroxyethyl)-[1,3]-dioxolane (VI, 0.42 g,0.59 mmol) and dry triethylamine (300 mg, 2.9 mmol) in 50 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.25 g, 1.4 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×25 mL), brine (25mL), and dried over anhydrous MgSO₄. The solvent was evaporated toafford 0.43 g of pale oil. The crude product was used in the followingstep without further purification.

7. Synthesis of2,2-Didocosahexaenoyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DDha-K5-C2-DMA)

To the above crude material (VII, 0.43 g) under argon was added 15 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 40mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in0.31 g of the product DDha-K5-C2-DMA as yellowish oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.25-5.45 (24H, m, 12×CH═CH), 4.05-4.17 (2H, m, 2×OCH), 3.50(1H, t, OCH), 2.87-3.15 (2H, br., NCH₂) 2.73-2.87 (20H, m,10×C═C—CH₂—C═C), 2.65 (6H, s, br, 2×NCH₃), 2.06 (8H, m, 4×allylic CH₂),2.0-2.2 (2H, m, CH₂), 1.75-1.95 (2H, m, CH₂), 1.3-1.65 (8H, m, 4×CH₂),0.90 (6H, t, 2×CH₃) ppm.

Example 26: Synthesis of Dilinoleyl 2-(2-Dimethylaminoethyl)-malonate(DLin-MAL-C2-DMA)

1. Synthesis of Dilinoleyl Malonate (II)

To a solution of linoleyl alcohol (I, 5.0 g, 19 mmol) in anhydrousCH₂Cl₂ (70 mL) was added dropwise malonyl dichloride (1.36 g, 9.3 mmol)under argon at 0-5° C. The resulting mixture was stirred at roomtemperature for 6 hours. The mixture was diluted with 50 mL of CH₂Cl₂.The organic phase was washed with water (3×75 mL), brine (75 mL) anddried over anhydrous Na₂SO₄. Evaporation of the solvent gave a brownishoily residual (5.8 g). The crude product was purified by columnchromatography on silica gel (230-400 mesh, 200 mL) with 0-4% ethylacetate gradient in hexanes as eluent. This afforded 3.1 g (55%) of pureII as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8, m,4×CH═CH), 4.13 (4H, t, 2×OCH₂), 3.35 (2H, s, CO—CH₂—CO), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.05 (8H, q, 4×allylic CH₂), 1.55-1.65 (4H, m, CH₂),1.2-1.4 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

2. Synthesis of Dilinoleyl 2-(2-Dimethylaminoethyl)-malonate(DLin-MAL-C2-DMA)

To a suspension of NaH (0.17 g, 60%, 4.1 mmol) in anhydrous benzene (40mL) was added dilinoleyl malonate (II, 0.50 g, 0.83 mmol) under argon.The resulting suspension was stirred at room temperature 60 min. To theresulting mixture was added N,N-dimethylamimoethyl chloridehydrochloride (0.12 g, 0.83 mmol) in one portion, and the resultingmixture was refluxed under argon for 2 days. The organic phase waswashed with water (3×20 mL), brine (2×25 mL) and dried over anhydrousNa₂SO₄. Evaporation of the solvent gave a pale oily residual (0.50 g).Column chromatography on silica gel (230-400 mesh, 40 mL) with 0-4%methanol in dichloromethane as eluent resulted in 0.13 g of the productDLin-MAL-C2-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.40 (8, m,4×CH═CH), 4.05-4.20 (4H, m, 2×OCH₂), 3.47 (1H, t, CO—CH—CO), 2.75 (4H,t, 2×C═C—CH₂—C═C), 2.35-2.9 (6H, br, 2×NCH₃), 2.15-2.35 (2H, br, NCH₂),2.05 (8H, q, 4×allylic CH₂), 1.55-1.65 (4H, m, CH₂), 1.2-1.45 (32H, m),0.90 (6H, t, 2×CH₃) ppm.

Example 27: Synthesis of Dilinoleyl 2-(2-Dimethylaminoethyl)-malonate(TetraLin-MAL-C2-DMA)

This compound was synthesized as pale oil in a similar manner to that inExample 26, where linoleyl alcohol was replaced by dilinoleyl methanol.¹H NMR (400 MHz, CDCl₃) δ: 5.15-5.50 (16, m, 8×CH═CH), 4.89 (2H,quintet), 3.46 (1H, t, CO—CH—CO), 3.08-3.2 (2H, m), 2.8-2.85 (6H, 2 s),2.78 (8H, t, 4×C═C—CH₂—C═C), 2.35-2.48 (2H, br, NCH₂), 2.05 (16H, q,8×allylic CH₂), 1.45-1.65 (8H, m, CH₂), 1.2-1.45 (64H, m), 0.90 (12H, t,2×CH₃) ppm.

Example 28: Synthesis of 4-Dimethylamino-butyric acid1-octadeca-6,9,12-trienyl-nonadeca-7,10,13-trienyl ester (005-14)

Compounds 005-8 to 005-12 were synthesized in a similar manner to thatin Example 19.

Under an argon atmosphere, to a round-bottom flask charged withDLen(γ)-MeOH (005-12, 262 mg, 0.5 mmol), 4-dimethylaminobutyric acidhydrochloride (101 mg, 0.6 mmol) and 4-(dimethylamino)pyridine (13 mg)in dichloromethane (5 mL) was added dicyclohexylcarbodiimide (134 mg).After the mixture is stirred for 16 hr at ambient temperature, thesolvent was evaporated and the residue was taken in diethyl ether. Thewhite precipitate is discarded by filtration. The filtrate wasconcentrated to dryness (0.4 g oil). The residue was purified by columnchromatography on silica gel (230-400 mesh, 50 mL) eluted with 2% to 3%of methanol in dichloromethane. Fractions containing the pure productwere combined and concentrated. The residue was passed through a layerof silica gel (2 mm) washed with hexanes (6 mL). The filtrate was thenconcentrated and dried in high vacuum for 1 h. This gave 166 mg (0.26mmol, 53%) of 005-14 as clear slightly yellow oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.41-5.26 (m, 12H, CH═CH), 4.83 (quintet, J=6 Hz, 1H), 2.77(t-like, J=5.2 Hz, 8H), 2.29 (t, J=7.6 Hz, 2H), 2.25 (t, J=7.6, 211),2.18 (s, 6H), 2.02 (q-like, J=6.8 Hz, 8H), 1.75 (quintet-like, J=7.6 Hz,2H), 1.48 (m, 4H), 1.37-1.20 (m, 24H), 0.86 (t, J=6.8 Hz, 6H) ppm.

Example 29: Synthesis of 5-Dimethylamino-pentanoic acid1-octadeca-6,9,12-trienyl-nonadeca-7,10,13-trienyl ester (005-23)

Step 1, 005-21

Under an argon atmosphere, to a round-bottom flask charged withDLen(γ)-MeOH (005-12, 262 mg, 0.5 mmol), 5-bromovaleric acid (181 mg,1.0 mmol) and 4-(dimethylamino)pyridine (30 mg) in dichloromethane (10mL) was added dicyclohexylcarbodiimide (227 mg). After the mixture isstirred for 16 hr at ambient temperature, the solvent was evaporated andthe residue was taken in hexanes. The white precipitate was discarded byfiltration. The filtrate was concentrated to dryness. The residue waspurified by column chromatography on silica gel (230-400 mesh, 50 mL)eluted with acetate in hexanes (0-2%). Fractions containing the pureproduct were combined and concentrated. This gave 290 mg (0.42 mmol,84%) of 005-21 as slightly yellow oil.

Step 2, 005-23

To 005-21 (290 mg) was added dimethylamine (2M in THF, 10 mL). Thesolution was stirred at room temperature for 6 days. The excess amineand solvent was evaporated. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 50 mL) with methanol indichloromethane (1 to 3%). Fractions containing the product werecombined and concentrated. The residue oil was passed through a layer ofcelite and washed with hexanes (6 mL). The filtrate was thenconcentrated and dried in high vacuum for 2 h. This gave 204 mg (0.31mmol, 74%) of 005-23 as slightly yellow oil. ¹H NMR (400 MHz, CDCl₃) δ:5.43-5.30 (m, 12H, CH═CH), 4.84 (quintet, J=6 Hz, 1H), 2.77 (t-like,J=5.2 Hz, 8H), 2.39-2.28 (m, 4H), 2.28 (s, 6H), 2.06 (q-like, J=6.8 Hz,8H), 1.66 (quintet-like, J=7.2 Hz, 2H), 1.60-1.48 (m, 6H), 1.41-1.24 (m,24H), 0.90 (t, 6H, J=6.8 Hz) ppm.

Example 30: Synthesis of[2-(2,2-Di-octadeca-6,9,12-trienyl-[1,3]dioxolan-4-yl)-ethyl]-dimethylamine(005-31)

Step 1, 005-28

To a mixture of dilinolenyl (γ) methanol (005-12, 550 mg, 1.05 mmol) andanhydrous potassium carbonate (58 mg) in 25 mL of anhydrous CH2C12 wasadded pyridinium chlorochromate (PCC, 566 mg, 2.63 mmol, 2.5 equiv.).The resulting suspension was stirred at room temperature for 90 min.Ether (100 mL) was then added into the mixture, and the resulting brownsuspension was filtered through a pad of silica gel (150 mL). The silicagel pad was further washed with ether (3×50 mL). The ether filtrate andwashings were combined. Evaporation of the solvent gave 510 mg of anoily residue as a crude product. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with0-3% of ethyl acetate in hexanes. This gave 344 g (63%) of the titledproduct (005-28).

Step 2, 005-29

A mixture of 005-28 (344 mg, 0.66 mmol), 1,2,4-butanetriol (349 mg, 3.2mmol) and pyridinium p-toluenesulfonate (30 mg) in 50 mL of toluene washeated to reflux under argon overnight with a Dean-Stark tube to removewater. The resulting mixture was cooled to room temperature. The organicphase was washed with water (30 mL) (the butanetriol is not soluble intoluene, so just decant the solution and the triol was left behind),brine (30 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)eluted with 4% ethyl acetate in hexanes. Fractions containing the pureproduct were combined and concentrated. This afforded 337 mg (83%) ofpure 005-29 as colorless oil.

Step 3, 005-30

To a solution of 005-29 (337 mg, 0.55 mmol) and dry triethylamine (0.28mL, 2 mmol) in 30 mL of anhydrous CH₂Cl₂ was added methanesulfonylanhydride (310 mg, 1.78 mmol) under nitrogen. The resulting mixture wasstirred at room temperature overnight. The mixture was diluted with 30mL of CH₂Cl₂. The organic phase was washed with water (2×25 mL), brine(25 mL), and dried over anhydrous MgSO₄. The solvent was evaporated toafford 377 g of the desired product as colorless clear oil (99%). Theproduct was pure enough and was used in the following step withoutfurther purification.

Step 4, 005-31

To 005-30 (377 mg) under argon was added 15 mL of dimethylamine in THF(2.0 M). The resulting mixture was stirred at room temperature for 6days. An oily residual was obtained upon evaporation of the solvent.Column chromatography on silica gel (230-400 mesh, 40 mL) eluted with 3%methanol in dichloromethane. Fractions containing the pure product werecombined and concentrated to give 314 mg of the titled product (005-31)as clear pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.41-5.26 (m, 12H, CH═CH),4.06 (m, 1H), 4.01 (dd, 1H, J=7.5, 7.5 Hz), 3.45 (dd, 1H, J=7.5, 7.5Hz), 2.77 (t-like, J=5.6 Hz, 8H), 2.36 (m, 1H), 2.26 (m, 1H), 2.19 (s,6H), 2.02 (q-like, J=6.8 Hz, 8H), 1.78 (m, 1H), 1.67 (m, 1H), 1.60-1.51(m, 4H), 1.38-1.21 (m, 24H), 0.86 (t, 6H, J=6.8 Hz) ppm.

Example 31: Synthesis of 4-(2-Methyl-aziridin-1-yl)-butyric acid1-octadeca-9,12-dienyl-nonadeca-10,13-dienyl ester (005-18)

Step 1, 005-13

Under an argon atmosphere, to a round-bottom flask charged withDLin-MeOH (001-17, 528.9 mg), 4-bromobutyric acid (200 mg) and4-(dimethylamino)pyridine (25 mg) in dichloromethane (10 mL) was addeddicyclohexylcarbodiimide (268 mg). After the mixture is stirred for 16hr at ambient temperature, the solvent was evaporated and the residuewas taken up in diethyl ether. The white precipitate (DCU) was discardedby filtration. The filtrate was concentrated and the resulting residualoil was purified by column chromatography on silica gel (230-400 mesh,50 mL) eluted with 0 to 1% of ethyl acetate in hexanes. This gave 0.44 g(65%) of 005-13 as colorless oil.

Step 2, 005-18

A mixture of 005-13 (0.44 g, 0.65 mmol), 2-methylaziridine (148 mg, 2.6mmol, tech. 90%), Cs₂CO3 (2.6 mmol) and TBAI (2.4 mmol) in acetonitrile(10 mL) was stirred under Ar for 4 days. After the solvent was removed,to the residue was added hexanes and water. The two phases wereseparated, followed by extraction of the aqueous phase with hexanes(×2). The combined organic phase was dried over sodium sulfate andconcentrated to dryness. The resulting residual oil was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with 1%to 3% of methanol in dichloromethane. Fractions containing the productwere combined and concentrated (200 mg of oil). This was purified againby column chromatography on silica gel (230-400 mesh, 50 mL) eluted withgradient ethyl acetate in hexanes (5%-20%). Fractions containing thepure product were combined and concentrated. This gave 96 mg (33%) of005-18 as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H,CH═CH), 4.87 (quintet, J=6 Hz, 1H), 2.78 (t-like, J=6 Hz, 4H), 2.39(t-like, J=7.8 Hz, 2H), 2.26 (t-like, 2H), 2.06 (q-like, J=6.8 Hz, 8H),1.89 (quintet-like, J=7.2 Hz, 2H), 1.56-1.48 (m, 5H), 1.41-1.24 (m,38H), 1.18 (d, J=5.2 Hz, 3H), 0.90 (t, 6H, J=6.8 Hz) ppm.

Example 32: Synthesis of2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6S-C1-DMA)

8. Synthesis of Linoleyl Bromide (II)

A mixture of linoleyl methane sulfonate (26.6 g, 77.2 mmol) and lithiumbromide (30.5 g, 350 mmol) in acetone (350 mL) was stirred undernitrogen for two days. The resulting suspension was filtered and thesolid washed with acetone. The filtrate and wash were combined andsolvent evaporated. The resulting residual was treated with water (300mL) The aqueous phase was extracted with ether (3×150 mL). The combinedether phase was washed with water (200 mL), brine (200 mL) and driedover anhydrous Na₂SO₄. The solvent was evaporated to afford 29.8 g ofyellowish oil. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 700 mL) eluted with hexanes. This gave 20.8g (82%) of linoleyl bromide (II).

9. Synthesis of Dilinoleylmethyl Formate (III)

To a suspension of Mg turnings (1.64 g, 67.4 mmol) with one crystal ofiodine in 500 mL of anhydrous ether under nitrogen was added a solutionof linoleyl bromide (II, 18.5 g, 56.1 mmol) in 250 mL of anhydrous etherat room temperature. The resulting mixture was refluxed under nitrogenovernight. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added dropwise ethyl formate (4.24 g, 57.2mmol). Upon addition, the mixture was stirred at room temperatureovernight. The mixture was treated with 10% H₂SO₄ aqueous solution (250mL). The ether phase was separated and aqueous phase extracted withether (150 mL). The combined organic phase was washed with water (400mL), brine (300 mL), and then dried over anhydrous Na₂SO₄. Evaporationof the solvent gave 17.8 g of yellowish oil as a crude product (III).The crude product was used directly in the following step withoutfurther purification.

10. Synthesis of Dilinoleyl Methanol (IV)

The above crude dilinoleylmethyl formate (III, 17.8 g) and KOH (3.75 g)were stirred in 85% EtOH at room temperature under nitrogen overnight.Upon completion of the reaction, most of the solvent was evaporated. Theresulting mixture was poured into 150 mL of 5% HCl solution. The aqueousphase was extracted with ether (2×150 mL). The combined ether extractwas washed with water (2×100 mL), brine (100 mL), and dried overanhydrous Na₂SO₄. Evaporation of the solvent gave 20.0 of dilinoleylmethanol (IV) as yellowish oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 700 mL) eluted with 0-5%ethyl acetate gradient in hexanes. This gave 9.6 g of dilinoleylmethanol (IV).

11. Synthesis of Dilinoleyl Ketone (V)

To a mixture of dilinoleyl methanol (4.0 g, 7.2 mmol) and anhydrouspotassium carbonate (0.4 g) in 100 mL of CH₂Cl₂ was added pyridiniumchlorochromate (PCC, 4.0 g, 19 mmol). The resulting suspension wasstirred at room temperature for 2 hours. Ether (300 mL) was then addedinto the mixture, and the resulting brown suspension was filteredthrough a pad of silica gel (150 mL). The silica gel pad was furtherwashed with ether (3×75 mL). The ether filtrate and washes werecombined. Evaporation of the solvent gave 5.1 g of an oily residual as acrude product. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 200 mL) eluted with 0-4% ethyl acetate inhexanes. This afforded 3.0 g (79%) of dilinoleyl ketone (V).

12. Synthesis of 2,2-Dilinoleyl-5-hydroxymethyl)-[1,3]-dioxane (VI)

A mixture of dilinoleyl ketone (V, 1.05 g, 2.0 mmol),2-hydroxymethyl-1,3-propanediol (490 mg, 4.2 mmol) and pyridiniump-toluenesulfonate (100 mg, 0.4 mmol) in 150 mL of toluene was refluxedunder nitrogen overnight with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×100 mL), brine (100 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (1.2 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 100 mL) with 0-5% methanol gradient in dichloromethane aseluent. This afforded 0.93 g of pure VI as pale oil.

13. Synthesis of 2,2-Dilinoleyl-5-methanesulfonylmethyl-[1,3]-dioxane(VII)

To a solution of 2,2-dilinoleyl-5-hydroxymethyl-[1,3]-dioxane (VI, 0.93g, 1.5 mmol) and dry triethylamine (290 mg, 2.9 mmol) in 50 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (400 mg, 2.3 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×75 mL), brine (75mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 1.0 g of pale oil. The crude product was used in the followingstep without further purification.

14. Synthesis of 2,2-Dilinoleyl-5-dimethylaminomethyl)-[1,3]-dioxane(DLin-K6S-C1-DMA)

To the above crude material (VII, 1.0 g) under nitrogen was added 20 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 7 days. An oily residual was obtained uponevaporation of the solvent. Column chromatography on silica gel (230-400mesh, 100 mL) with 0-3% methanol gradient in chloroform as eluentresulted in 150 mg of the product DLin-K6S-C1-DMA as pale oil.

¹H NMR (400 MHz, CDCl₃) δ: 5.24-5.51 (8, m, 4×CH═CH), 4.04 (2H, dd,2×OCH)), 3.75 (2H, dd OCH), 2.7-2.9 (2H, br, NCH₂), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.57 (6H, s, 2×NCH₃), 1.95-2.17 (9H, q, 4×allylic CH₂and CH), 1.67-1.95 (2H, m, CH₂), 1.54-1.65 (4H, m, 2×CH₂), 1.22-1.45(32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 33: Synthesis of2,2-Dilinoleyl-5-dimethylaminobutyl-[1,3]-dioxane (DLin-K6S-C4-DMA)

This compound was synthesized as pale oil in a similar manner to that inExample 32, where 2-hydroxymethyl-1,3-propanediol was replaced with2-hydroxybutyl-1,3-propanediol. ¹H NMR (400 MHz, CDCl₃) δ: 5.24-5.45 (8,m, 4×CH═CH), 3.79 (2H, dd, 2×OCH)), 3.50 (2H, dd OCH), 2.76 (4H, t,2×C═C—CH₂—C═C), 2.37 (2H, t, NCH₂), 2.31 (6H, s, 2×NCH₃), 2.04 (8H, q,4×allylic CH₂), 1.63-1.90 (3H, m,), 1.45-1.62 (4H, m, 2×CH₂), 1.22-1.45(36H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 34: Synthesis of2,2-Dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxane (DLin-K6S-C2-DMA)

This compound was synthesized as pale oil in a similar manner to that inExample 32, where 2-hydroxymethyl-1,3-propanediol was replaced with2-hydroxyethyl-1,3-propanediol. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8,m, 4×CH═CH), 3.87 (2H, dd, 2×OCH)), 3.55 (2H, dd OCH), 2.75 (4H, t,2×C═C—CH₂—C═C), 2.45-2.60 (2H, br, NCH₂), 2.40 (6H, s, 2×NCH₃), 2.03(8H, q, 4×allylic CH₂), 1.73-1.86 (1H, m), 1.56-1.72 (6H, m, 2×CH₂),1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 35: Synthesis of2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane (DLin-K6A-C2-DMA)

5. Synthesis of 1,3,5-Pentanetriol (II)

Diethyl 3-hydroxyglutarate (I, 1.0 g, 4.9 mmol) in anhydrous THF (10 mL)was added dropwise to a suspension of LiAlH₄ in anhydrous THF (110 mL)under nitrogen with a cold water bath. Upon addition, the bath wasremoved and the suspension was stirred at room temperature for 2 days.The resulting mixture was quenched by adding 13 mL of brine very slowlywith an ice-water bath. A white suspension was resulted, and the mixturewas stirred at room temperature overnight. The solid was filtered, andwashed with THF. The filtrate and wash were combined, and solventevaporated to give 0.70 g of pale oil. Column chromatography of thecrude product (230-400 mesh SiO₂, 100 mL, 0-12% methanol gradient inchloroform) afforded 0.54 g of II as colourless oil.

6. Synthesis of 2,2-Dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxane (IV)

A mixture of dilinoleyl ketone (III, 0.80 g, 1.5 mmol),1,3,5-pentanetriol (II, 0.54 g, 4.5 mmol) and pyridiniump-toluenesulfonate (60 mg, 0.24 mmol) in 150 mL of toluene was refluxedunder nitrogen overnight with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×75 mL), brine (75 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (1.1 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 75 mL) with 0-3% methanol gradient in dichloromethane aseluent. This afforded 0.75 g (79%) of pure IV as colourless oil.

7. Synthesis of 2,2-Dilinoleyl-4-(2-methanesulfonylethyl)-[1,3]-dioxane(V)

To a solution of 2,2-dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxane (IV,0.75 g, 1.2 mmol) and dry triethylamine (0.58 g, 5.7 mmol) in 40 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.50 g, 2.9 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×50 mL), brine (50mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 0.80 g of pale oil as a crude product. The crude product was usedin the following step without further purification.

8. Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane(DLin-K6A-C2-DMA)

To the above crude material (V, 0.80 g) under nitrogen was added 15 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 6 days. The solid was filtered. An oily residualwas obtained upon evaporation of the solvent. Column chromatography onsilica gel (230-400 mesh, 100 mL) with 0-6% methanol gradient indichloromethane as eluent resulted in 0.70 g of the productDLin-K6A-C2-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.28-5.45 (8, m,4×CH═CH), 3.85-4.0 (2H, m, 2×OCH), 3.78 (1H, dd, OCH), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.55-2.90 (2H, br, NCH₂), 2.47 (6H, s, 2×NCH₃), 2.05(8H, q, 4×allylic CH₂), 1.65-1.90 (4H, m, CH₂), 1.47-1.65 (4H, m, CH₂),1.1-1.65 (36H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 36: Synthesis of2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxane (DLin-K6A-C3-DMA)

5. Synthesis of 1,3,6-Hexanetriol (II)

Diethyl β-ketoadipate (I, 1.86 g, 8.6 mmol) was added dropwise to asuspension of LiAlH₄ in anhydrous THF (90 mL) under argon with anice-water bath. Upon addition, the bath was removed and the suspensionwas stirred at room temperature overnight. The resulting mixture wasquenched by adding 10 mL of brine very slowly with an ice-water bath. Awhite suspension was resulted, and the mixture was stirred at roomtemperature overnight. The solid was filtered, and washed with THFfollowed by EtOH (2×50 mL). The filtrate and wash were combined, andsolvent evaporated to give 0.90 g of pale oil. Column chromatography ofthe crude product (230-400 mesh SiO₂, 100 mL, 0-10% methanol gradient indichloromethane) afforded 0.70 g of II as colourless oil.

6. Synthesis of 2,2-Dilinoleyl-4-(3-hydroxypropyl)-[1,3]-dioxane (IV)

A mixture of dilinoleyl ketone (III, 1.80 g, 3.4 mmol),1,3,6-hexanetriol (II, 0.50 g, 3.7 mmol) and pyridiniump-toluenesulfonate (100 mg, 0.40 mmol) in 120 mL of toluene was refluxedunder argon for 3 hours with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×50 mL), brine (50 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in pale oil (2.0 g). Thecrude product was purified by column chromatography on silica gel(230-400 mesh, 50 mL) with 0-3% methanol gradient in dichloromethane aseluent. This afforded 0.90 g (41%) of pure IV as colourless oil.

7. Synthesis of 2,2-Dilinoleyl-4-(3-methanesulfonylpropyl)-[1,3]-dioxane(V)

To a solution of 2,2-dilinoleyl-4-(3-hydroxypropyl)-[1,3]-dioxane (IV,0.97 g, 1.5 mmol) and dry triethylamine (0.44 g, 4.3 mmol) in 60 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.60 g, 3.5 mmol)under argon. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×30 mL), brine (30mL), and dried over anhydrous MgSO₄. The solvent was evaporated toafford 1.1 g of pale oil as a crude product. The crude product was usedin the following step without further purification.

8. Synthesis of 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxane(DLin-K6A-C3-DMA)

To the above crude material (V, 1.1 g) under argon was added 20 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 5 days. The solid was filtered. An oily residual wasobtained upon evaporation of the solvent. Column chromatography onsilica gel (230-400 mesh, 40 mL) with 0-7% methanol gradient indichloromethane as eluent resulted in 0.85 g of the productDLin-K6A-C3-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8, m,4×CH═CH), 3.7-4.0 (3H, m, 3×OCH), 2.77 (4H, t, 2×C═C—CH₂—C═C), 2.5-2.8(2H, br, NCH₂), 2.5 (6H, s, 2×NCH₃), 2.05 (8H, q, 4×allylic CH₂),1.65-1.90 (4H, m, 2×CH₂), 1.40-1.65 (4H, m, 2×CH₂), 1.1-1.65 (38H, m),0.90 (6H, t, 2×CH₃) ppm.

Example 37: Synthesis of2,2-Diarachidonyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DAra-K5-C2-DMA)

8. Synthesis of Arachidonyl Bromide (II)

A mixture of arachidonyl methane sulfonate (1.0 g, 2.7 mmol) andmagnesium bromide (2.2 g, 12 mmol) in anhydrous ether (40 mL) wasstirred under argon for two days. The resulting suspension was filteredand the solid washed with ether (2×10 mL). The filtrate and wash werecombined and solvent evaporated. The resulting residual was treated withhexanes (50 mL). The solid was filtered and solvent evaporated resultingin an oily residual. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 30 mL) eluted with hexanes.This gave 1 g of arachidonyl bromide (II) as colourless oil.

9. Synthesis of Diarachidonylmethyl Formate (III)

To a solution of arachidonyl bromide (II, 1 g, 3 mmol) in anhydrousether (30 mL) was added Mg turnings (78 mg, 3.2 mmol) followed by onecrystal of iodine. The resulting mixture was refluxed under nitrogen for10 hours. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added ethyl formate (0.25 mL), and theresulting mixture was stirred at room temperature overnight. To themixture was added 20 mL of 10% H₂SO₄ aqueous solution. The ether phasewas separated and aqueous phase extracted with ether (30 mL) Thecombined organic phase was washed with water (2×25 mL), brine (25 mL),and then dried over anhydrous Na₂SO₄. Evaporation of the solvent gave1.1 g of pale oil as a crude product (III). The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)eluted with 0-3% ethyl acetate gradient in hexanes. This afforded 0.43 g(40%) of diarachidonylmethyl formate (III) as pale oil.

10. Synthesis of Diarachidonyl Methanol (IV)

The above diarachidonylmethyl formate (III, 0.43 g, 0.71 mmol) and KOH(100 mg) were stirred in 95% EtOH (20 mL) at room temperature undernitrogen overnight. Upon completion of the reaction, most of the solventwas evaporated. The resulting mixture was treated with 20 mL of 2M HClsolution. The aqueous phase was extracted with ether (2×30 mL). Thecombined ether extract was washed with water (2×25 mL), brine (25 mL),and dried over anhydrous Na₂SO₄. Evaporation of the solvent gave 0.44 gof IV as pale oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 40 mL) eluted with 0-5%ethyl acetate gradient in hexanes. This gave 0.41 g of diarachidonylmethanol (IV) as colourless oil.

11. Synthesis of Diarachidonyl Ketone (V)

To a mixture of diarachidonyl methanol (IV, 0.41 g, 0.71 mmol) andanhydrous potassium carbonate (0.05 g) in 10 mL of CH₂Cl₂ was addedpyridinium chlorochromate (PCC, 0.50 g, 2.3 mmol). The resultingsuspension was stirred at room temperature for 90 mins. Ether (50 mL)was then added into the mixture, and the resulting brown suspension wasfiltered through a pad of floresil (30 mL) The pad was further washedwith ether (3×30 mL). The ether filtrate and washes were combined.Evaporation of the solvent gave 0.40 g of an oily residual as a crudeproduct. The crude product was purified by column chromatography onsilica gel (230-400 mesh, 10 mL) eluted with 0-3% ether in hexanes. Thisafforded 0.30 g (75%) of diarachidonyl ketone (V). ¹H NMR (400 MHz,CDCl₃) δ: 5.3-5.5 (16H, m, 8×CH═CH), 2.82 (12H, t, 6×C═C—CH₂—C═C), 2.40(4H, t, 2×CO—CH₂), 2.08 (8H, m, 4×allylic CH₂), 1.25-1.65 (20H, m), 0.90(6H, t, 2×CH₃) ppm.

12. Synthesis of 2,2-Diarachidonyl-4-(2-hydroxyethyl)-[1,3]-dioxolane(VI)

A mixture of diarachidonyl ketone (V, 0.30 g, 0.52 mmol),1,2,4-butanetriol (0.25 g, 2.4 mmol) and pyridinium p-toluenesulfonate(20 mg) in 60 mL of toluene was refluxed under argon overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (30 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)with 0-2% methanol in dichloromethane as eluent. This afforded 0.29 g(84%) of pure VI as pale oil.

13. Synthesis of2,2-Diarachidonyl-4-(2-methanesulfonylethyl)[1,3]-dioxolane (VII)

To a solution of 2,2-diarachidonyl-4-(2-hydroxyethyl)-[1,3]-dioxolane(VI, 0.29 g, 0.43 mmol) and dry triethylamine (254 mg, 2.5 mmol) in 20mL of anhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.20 g, 1.1mmol) under nitrogen. The resulting mixture was stirred at roomtemperature overnight. The mixture was diluted with 30 mL of CH₂Cl₂. Theorganic phase was washed with water (2×25 mL), brine (25 mL), and driedover anhydrous MgSO₄. The solvent was evaporated to afford 0.30 g ofpale oil. The crude product was used in the following step withoutfurther purification.

14. Synthesis of2,2-Diarachidonyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DAra-K5-C2-DMA)

To the above crude material (VII, 0.30 g) under argon was added 15 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 40mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in0.18 g of the product DAra-K5-C2-DMA as pale oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.3-5.5 (16H, m, 8×CH═CH), 4.0-4.17 (2H, m, 2×OCH), 3.49 (1H,t, OCH), 2.65-2.85 (14H, m, 6×C═C—CH₂—C═C, NCH₂), 2.55 (6H, s, br,2×NCH₃), 2.06 (8H, m, 4×allylic CH₂), 1.80-1.92 (2H, m, CH₂), 1.4-1.75(4H, m, 2×CH₂), 1.22-1.45 (20H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 38: Synthesis of2,2-Didocosahexaenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DDha-K5-C2-DMA)

8. Synthesis of Docosahexaenoyl Bromide (II)

A mixture of docosahexaenoyl methane sulfonate (2.0 g, 5.1 mmol) andmagnesium bromide (4.3 g, 23 mmol) in anhydrous ether (100 mL) wasstirred under argon overnight. The resulting suspension was filtered andthe solid washed with ether (2×30 mL) The filtrate and wash werecombined and solvent evaporated. The resulting residual was purified bycolumn chromatography on silica gel (230-400 mesh, 40 mL) eluted withhexanes. This gave 2.2 g of docosahexaenoyl bromide (II) as colourlessoil.

9. Synthesis of Didocosahexaenoylmethyl Formate (III)

To a solution of docosahexaenoyl bromide (II, 2.2 g, 6.0 mmol) inanhydrous ether (60 mL) was added Mg turnings (145 mg, 6.0 mmol)followed by one crystal of iodine. The resulting mixture was refluxedunder argon for 5 hours. The mixture was cooled to room temperature. Tothe cloudy mixture under argon was added ethyl formate (0.50 mL), andthe resulting mixture was stirred at room temperature overnight. To themixture was added 40 mL of 5% H₂SO₄ aqueous solution. The ether phasewas separated and aqueous phase extracted with ether (50 mL). Thecombined organic phase was washed with water (2×50 mL), brine (50 mL),and then dried over anhydrous MgSO₄. Evaporation of the solvent gave 2.3g of yellowish oil as a crude product (III). The crude product waspurified by column chromatography on silica gel (230-400 mesh, 50 mL)eluted with 0-7% ethyl acetate gradient in hexanes. This afforded 1.38 g(65%) of didocosahexaenoylmethyl formate (III) as pale oil.

10. Synthesis of Didocosahexaenoyl Methanol (IV)

The above didocosahexaenoylmethyl formate (III, 1.38 g, 2.1 mmol) andKOH (300 mg) were stirred in 90% EtOH (70 mL) at room temperature undernitrogen for 90 min. Upon completion of the reaction, most of thesolvent was evaporated. The resulting mixture was treated with 60 mL of2M HCl solution. The aqueous phase was extracted with ether (2×75 mL).The combined ether extract was washed with water (2×50 mL), brine (50mL), and dried over anhydrous MgSO₄. Evaporation of the solvent gave1.18 g of crude IV as yellowish oil. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with0-6% ethyl acetate gradient in hexanes. This gave 1.0 g ofdidocosahexaenoyl methanol (IV) as colourless oil.

11. Synthesis of Didocosahexaenoyl Ketone (V)

To a mixture of didocosahexaenoyl methanol (IV, 1.2 g, 1.9 mmol) andanhydrous potassium carbonate (0.1 g) in 30 mL of CH₂Cl₂ was addedpyridinium chlorochromate (PCC, 1.05 g, 4.8 mmol). The resultingsuspension was stirred at room temperature for 2 hours. Ether (120 mL)was then added into the mixture, and the resulting brown suspension wasfiltered through a pad of silica gel (75 mL). The pad was further washedwith ether (3×75 mL). The ether filtrate and washes were combined.Evaporation of the solvent gave 1.3 g of an oily residual as a crudeproduct. The crude product was purified by column chromatography onsilica gel (230-400 mesh, 40 mL) eluted with 0-3% ethyl acetate inhexanes. This afforded 0.83 g (69%) of didocosahexaenoyl ketone (V).

12. Synthesis of 2,2-Didocosahexaenoyl-4-(2-hydroxyethyl)[1,3]-dioxolane(VI)

A mixture of diarachidonyl ketone (V, 0.43 g, 0.69 mmol),1,2,4-butanetriol (0.35 g, 3.3 mmol) and pyridinium p-toluenesulfonate(50 mg) in 75 mL of toluene was refluxed under argon overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (30 mL), and dried over anhydrous MgSO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)with 0-2% methanol in dichloromethane as eluent. This afforded 0.43 g(95%) of pure VI as pale oil.

13. Synthesis of2,2-Didocosahexaenoyl-4-(2-methanesulfonylethyl)-[1,3]-dioxolane (VII)

To a solution of 2,2-didocosahexaenoyl-4-(2-hydroxyethyl)[1,3]-dioxolane(VI, 0.42 g, 0.59 mmol) and dry triethylamine (300 mg, 2.9 mmol) in 50mL of anhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.25 g, 1.4mmol) under nitrogen. The resulting mixture was stirred at roomtemperature overnight. The organic phase was washed with water (2×25mL), brine (25 mL), and dried over anhydrous MgSO₄. The solvent wasevaporated to afford 0.43 g of pale oil. The crude product was used inthe following step without further purification.

14. Synthesis of2,2-Didocosahexaenoyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DDha-K5-C2-DMA)

To the above crude material (VII, 0.43 g) under argon was added 15 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 40mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in0.31 g of the product DDha-K5-C2-DMA as yellowish oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.25-5.45 (24H, m, 12×CH═CH), 4.05-4.17 (2H, m, 2×OCH), 3.50(1H, t, OCH), 2.87-3.15 (2H, br., NCH₂) 2.73-2.87 (20H, m,10×C═C—CH₂—C═C), 2.65 (6H, s, br, 2×NCH₃), 2.06 (8H, m, 4×allylic CH₂),2.0-2.2 (2H, m, CH₂), 1.75-1.95 (2H, m, CH₂), 1.3-1.65 (8H, m, 4×CH₂),0.90 (6H, t, 2×CH₃) ppm.

Example 39: Synthesis of 4-Dimethylamino-butyric acid1-octadeca-6,9,12-trienyl-nonadeca-7,10,13-trienyl ester (005-14)

Under an argon atmosphere, to a round-bottom flask charged withDLen(γ)-MeOH (005-12, 262 mg, 0.5 mmol), 4-dimethylaminobutyric acidhydrochloride (101 mg, 0.6 mmol) and 4-(dimethylamino)pyridine (13 mg)in dichloromethane (5 mL) was added dicyclohexylcarbodiimide (134 mg).After the mixture is stirred for 16 hr at ambient temperature, thesolvent was evaporated and the residue was taken in diethyl ether. Thewhite precipitate is discarded by filtration. The filtrate wasconcentrated to dryness (0.4 g oil). The residue was purified by columnchromatography on silica gel (230-400 mesh, 50 mL) eluted with 2% to 3%of methanol in dichloromethane. Fractions containing the pure productwere combined and concentrated. The residue was passed through a layerof silica gel (2 mm) washed with hexanes (6 mL). The filtrate was thenconcentrated and dried in high vacuum for 1 h. This gave 166 mg (0.26mmol, 53%) of 005-14 as clear slightly yellow oil. ¹H NMR (400 MHz,CDCl₃) δ: 5.41-5.26 (m, 12H, CH═CH), 4.83 (quintet, J=6 Hz, 1H), 2.77(t-like, J=5.2 Hz, 8H), 2.29 (t, J=7.6 Hz, 2H), 2.25 (t, J=7.6, 2H),2.18 (s, 6H), 2.02 (q-like, J=6.8 Hz, 8H), 1.75 (quintet-like, J=7.6 Hz,2H), 1.48 (m, 4H), 1.37-1.20 (m, 24H), 0.86 (t, J=6.8 Hz, 6H) ppm.

Example 40: Synthesis of 5-Dimethylamino-pentanoic acid1-octadeca-6,9,12-trienyl-nonadeca-7,10,13-trienyl ester (005-23)

Step 1, 005-21

Under an argon atmosphere, to a round-bottom flask charged withDLen(γ)-MeOH (005-12, 262 mg, 0.5 mmol), 5-bromovaleric acid (181 mg,1.0 mmol) and 4-(dimethylamino)pyridine (30 mg) in dichloromethane (10mL) was added dicyclohexylcarbodiimide (227 mg). After the mixture isstirred for 16 hr at ambient temperature, the solvent was evaporated andthe residue was taken in hexanes. The white precipitate was discarded byfiltration. The filtrate was concentrated to dryness. The residue waspurified by column chromatography on silica gel (230-400 mesh, 50 mL)eluted with acetate in hexanes (0-2%). Fractions containing the pureproduct were combined and concentrated. This gave 290 mg (0.42 mmol,84%) of 005-21 as slightly yellow oil.

Step 2, 005-23

To 005-21 (290 mg) was added dimethylamine (2M in THF, 10 mL). Thesolution was stirred at room temperature for 6 days. The excess amineand solvent was evaporated. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 50 mL) with methanol indichloromethane (1 to 3%). Fractions containing the product werecombined and concentrated. The residue oil was passed through a layer ofcelite and washed with hexanes (6 mL). The filtrate was thenconcentrated and dried in high vacuum for 2 h. This gave 204 mg (0.31mmol, 74%) of 005-23 as slightly yellow oil. ¹H NMR (400 MHz, CDCl₃) δ:5.43-5.30 (m, 12H, CH═CH), 4.84 (quintet, J=6 Hz, 1H), 2.77 (t-like,J=5.2 Hz, 8H), 2.39-2.28 (m, 4H), 2.28 (s, 6H), 2.06 (q-like, J=6.8 Hz,8H), 1.66 (quintet-like, J=7.2 Hz, 2H), 1.60-1.48 (m, 6H), 1.41-1.24 (m,24H), 0.90 (t, 6H, J=6.8 Hz) ppm.

Example 41: Synthesis of[2-(2,2-Di-octadeca-6,9,12-trienyl-[1,3]dioxolan-4-yl)-ethyl]-dimethylamine(005-31)

Step 1, 005-28

To a mixture of dilinolenyl (γ) methanol (005-12, 550 mg, 1.05 mmol) andanhydrous potassium carbonate (58 mg) in 25 mL of anhydrous CH2Cl2 wasadded pyridinium chlorochromate (PCC, 566 mg, 2.63 mmol, 2.5 equiv.).The resulting suspension was stirred at room temperature for 90 min.Ether (100 mL) was then added into the mixture, and the resulting brownsuspension was filtered through a pad of silica gel (150 mL). The silicagel pad was further washed with ether (3×50 mL). The ether filtrate andwashings were combined. Evaporation of the solvent gave 510 mg of anoily residue as a crude product. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with0-3% of ethyl acetate in hexanes. This gave 344 g (63%) of the titledproduct (005-28).

Step 2, 005-29

A mixture of 005-28 (344 mg, 0.66 mmol), 1,2,4-butanetriol (349 mg, 3.2mmol) and pyridinium p-toluenesulfonate (30 mg) in 50 mL of toluene washeated to reflux under argon overnight with a Dean-Stark tube to removewater. The resulting mixture was cooled to room temperature. The organicphase was washed with water (30 mL) (the butanetriol is not soluble intoluene, so just decant the solution and the triol was left behind),brine (30 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual. The crude product waspurified by column chromatography on silica gel (230-400 mesh, 40 mL)eluted with 4% ethyl acetate in hexanes. Fractions containing the pureproduct were combined and concentrated. This afforded 337 mg (83%) ofpure 005-29 as colorless oil.

Step 3, 005-30

To a solution of 005-29 (337 mg, 0.55 mmol) and dry triethylamine (0.28mL, 2 mmol) in 30 mL of anhydrous CH₂Cl₂ was added methanesulfonylanhydride (310 mg, 1.78 mmol) under nitrogen. The resulting mixture wasstirred at room temperature overnight. The mixture was diluted with 30mL of CH₂Cl₂. The organic phase was washed with water (2×25 mL), brine(25 mL), and dried over anhydrous MgSO₄. The solvent was evaporated toafford 377 g of the desired product as colorless clear oil (99%). Theproduct was pure enough and was used in the following step withoutfurther purification.

Step 4, 005-31

To 005-30 (377 mg) under argon was added 15 mL of dimethylamine in THF(2.0 M). The resulting mixture was stirred at room temperature for 6days. An oily residual was obtained upon evaporation of the solvent.Column chromatography on silica gel (230-400 mesh, 40 mL) eluted with 3%methanol in dichloromethane. Fractions containing the pure product werecombined and concentrated to give 314 mg of the titled product (005-31)as clear pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.41-5.26 (m, 12H, CH═CH),4.06 (m, 1H), 4.01 (dd, 1H, J=7.5, 7.5 Hz), 3.45 (dd, 1H, J=7.5, 7.5Hz), 2.77 (t-like, J=5.6 Hz, 8H), 2.36 (m, 1H), 2.26 (m, 1H), 2.19 (s,6H), 2.02 (q-like, J=6.8 Hz, 8H), 1.78 (m, 1H), 1.67 (m, 1H), 1.60-1.51(m, 4H), 1.38-1.21 (m, 24H), 0.86 (t, 6H, J=6.8 Hz) ppm.

Example 42: Synthesis of 4-(2-Methyl-aziridin-1-yl)-butyric acid1-octadeca-9,12-dienyl-nonadeca-10,13-dienyl ester (005-18)

Step 1, 005-13

Under an argon atmosphere, to a round-bottom flask charged withDLin-MeOH (001-17, 528.9 mg), 4-bromobutyric acid (200 mg) and4-(dimethylamino)pyridine (25 mg) in dichloromethane (10 mL) was addeddicyclohexylcarbodiimide (268 mg). After the mixture is stirred for 16hr at ambient temperature, the solvent was evaporated and the residuewas taken up in diethyl ether. The white precipitate (DCU) was discardedby filtration. The filtrate was concentrated and the resulting residualoil was purified by column chromatography on silica gel (230-400 mesh,50 mL) eluted with 0 to 1% of ethyl acetate in hexanes. This gave 0.44 g(65%) of 005-13 as colorless oil.

Step 2, 005-18

A mixture of 005-13 (0.44 g, 0.65 mmol), 2-methylaziridine (148 mg, 2.6mmol, tech. 90%), Cs₂CO3 (2.6 mmol) and TBAI (2.4 mmol) in acetonitrile(10 mL) was stirred under Ar for 4 days. After the solvent was removed,to the residue was added hexanes and water. The two phases wereseparated, followed by extraction of the aqueous phase with hexanes(×2). The combined organic phase was dried over sodium sulfate andconcentrated to dryness. The resulting residual oil was purified bycolumn chromatography on silica gel (230-400 mesh, 50 mL) eluted with 1%to 3% of methanol in dichloromethane. Fractions containing the productwere combined and concentrated (200 mg of oil). This was purified againby column chromatography on silica gel (230-400 mesh, 50 mL) eluted withgradient ethyl acetate in hexanes (5%-20%). Fractions containing thepure product were combined and concentrated. This gave 96 mg (33%) of005-18 as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H,CH═CH), 4.87 (quintet, J=6 Hz, 1H), 2.78 (t-like, J=6 Hz, 4H), 2.39(t-like, J=7.8 Hz, 2H), 2.26 (t-like, 2H), 2.06 (q-like, J=6.8 Hz, 8H),1.89 (quintet-like, J=7.2 Hz, 2H), 1.56-1.48 (m, 5H), 1.41-1.24 (m,38H), 1.18 (d, J=5.2 Hz, 3H), 0.90 (t, 6H, J=6.8 Hz) ppm.

Example 43: Synthesis of 4-Dimethylamino-but-2-enoic acid1-octadeca-9,12-dienyl-nonadeca-10,13-dienyl ester (005-34)

Step 1, 005-32

Under an argon atmosphere, to a round-bottom flask charged withDLin-MeOH (001-17, 528.9 mg, 1 mmol), 4-bromocrotonic acid (330 mg, 2mmol) and 4-(dimethylamino)pyridine (49 mg) in dichloromethane (10 mL)was added dicyclohexylcarbodiimide (454 mg, 2.2 mmol). After the mixtureis stirred for 16 hr at ambient temperature, the precipitate was removedby filtration and the solid was washed with dichloromethane. To thefiltrate was added 4-bromocrotonic acid (165 mg),4-(dimethylamino)pyridine (15 mg) and finally dicyclohexylcarbodiimide(250 mg). After the mixture is stirred for 16 hr at ambient temperature,the solvent was evaporated and the residue was taken in hexanes. Thewhite precipitate (DCU) was discarded by filtration. The filtrate wasconcentrated and the resulting residue oil (587 mg) was used for thenext step without further purification.

Step 2, 005-34

To the crude 005-32 (587 mg) under argon was added 7 mL of dimethylaminein THF (2.0 M). The resulting mixture was stirred at room temperaturefor 3 days. An oily residual was obtained upon evaporation of thesolvent and was purified by column chromatography on silica gel (230-400mesh, 40 mL) eluted with dichloromethane 100 mL, 1% to 3% of methanol indichloromethane. Fractions containing the pure product were combined andconcentrated to give brownish oil (XD-005-34, 69 mg, 11% from DLin-MeOH,001-17). ¹H NMR (600 MHz, CDCl₃) δ: 6.92 (dt, J=6.2 Hz, 15.7 Hz, 1H),5.97 (d, J=15.7 Hz), 5.41-5.31 (8H, m, CH═CH), 4.93 (quintet, J=6.7 Hz,1H), 3.07 (dd, J=1.1 Hz, 6.2 Hz, 2H), 2.78 (t, J=6.9 Hz, 4H), 2.27 (s,6H), 2.05 (m, 8H), 1.58-1.52 (m, 4H), 1.39-1.24 (m, 36H), 0.90 (t, 6H,J=6.8 Hz) ppm.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

We claim:
 1. A lipid particle comprising mRNA as a therapeutic agent and a lipid having the structure

or a salt or isomer thereof, wherein: R₁ and R₂ are each independently for each occurrence optionally substituted C₁₀-C₃₀ alkenyl or optionally substituted C₁₀-C₃₀ alkynyl; R₃ is ω-aminoalkyls or ω-(substituted)aminoalkyls; and E is C(O)O; provided that when R₃ is 2-(dimethylamino)ethyl, R₁ and R₂ are not each linoleyl.
 2. The lipid particle of claim 1, wherein R₃ is ω-aminoalkyl.
 3. The lipid particle of claim 1, wherein R₃ is ω-(substituted)aminoalkyl.
 4. The lipid particle of claim 1, wherein R₃ is 2-(dimethylamino)ethyl, 3-(diisopropylamino)propyl, or 3-(N-ethyl-n-isopropylamino)-1-methylpropyl.
 5. The lipid particle of claim 1, wherein R₃ is alkylaminoalkyl or dialkylaminoalkyl.
 6. The lipid particle of claim 1, wherein R₁ and R₂ are each optionally substituted C₁₀-C₂₀ alkenyl.
 7. The lipid particle of claim 1, wherein R₁ and R₂ are each linoleyl.
 8. The lipid particle of claim 1, wherein the particle further comprises a neutral lipid and a lipid capable of reducing aggregation.
 9. The lipid particle of claim 8, wherein the lipid capable of reducing aggregation is a PEG-lipid.
 10. The lipid particle of claim 1, further comprising a neutral lipid, cholesterol, and a PEG lipid.
 11. The lipid particle of claim 10, wherein the lipid of formula XXXIII or a salt or isomer thereof, the neutral lipid, the cholesterol, and the PEG lipid are present in molar ratios of 20-70%:5-45%:20-55%:0.5-15%, respectively.
 12. A pharmaceutical composition comprising a lipid particle of claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent. 